Kidney-specific tumor vaccine directed against kidney tumor antigen g-250

ABSTRACT

This invention provides an anti-cancer immunogenic agent(s) (e.g. vaccines) that elicit an immune response specifically directed against renal cell cancers expressing a G250 antigenic marker. Preferred immunogenic agents comprise a chimeric molecule comprising a kidney cancer specific antigen (G250) attached to a granulocyte-macrophage colony stimulating factor (GM-CSF). The agents are useful in a wide variety of treatment modalities including, but not limited to protein vaccination, DNA vaccination, and adoptive immunotherapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/479,415, filed on Jun. 5, 2009, issued as U.S. Pat. No. 8,741,306,which is a divisional of U.S. application Ser. No. 09/783,708, filed onFeb. 13, 2001, issued as U.S. Pat. No. 7,572,891, which claims benefitof U.S. provisional applications U.S. Ser. No. 60/182,429, filed on Feb.14, 2000, and U.S. Ser. No. 60/182,636, filed on Feb. 15, 2000, each ofwhich are incorporated herein by reference in their entirety for allpurposes.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file-1459-3.TXT, created on Jun. 5,2014, 24,576 bytes, machine format IBM-PC, MS-Windows operating system,is hereby incorporated by reference in its entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

[Not Applicable]

FIELD OF THE INVENTION

This invention relates to the field of oncology. In particular thisinvention provides novel vaccines for use in the treatment of renal cellcancers.

BACKGROUND OF THE INVENTION

Renal cell carcinoma (RCC), also often identified as renal cancer,“hypernephroma”, or adenocarcinoma of the kidney accounts for about 85percent of all primary renal neoplasms. Approximately 25,000 new casesare diagnosed annually with 10,000 deaths in the United States.Unfortunately, the prognosis of patients with recurrent or metastaticrenal cell carcinoma remains poor. Chemotherapy and radiotherapy havelittle or no activity in this disease and there is no standardchemotherapeutic, hormonal, or immunologic program for recurrent ormetastatic renal cancer.

Commonly employed chemotherapy programs include the use of vinblastinesulfate, with or without the use of nitrosoureas. Interferons have beenused with very limited success. Interleukin 2 (Aldesleukin) is approvedfor treatment of selected patients with metastatic renal cell carcinoma.An overall response rate of 15 percent has been noted in 255 patients,but this has been accompanied by both severe adverse reactions an a fewtreatment-related deaths. Other treatment options for patients withadvanced disease are, at best, investigational.

SUMMARY OF THE INVENTION

This invention provides a novel approach to the treatment of renal cellcarcinomas. In particular this invention pertains to the discovery thata chimeric molecule comprising a granulocyte macrophage colonystimulating factor (GM-CSF) attached to a G250 kidney cancer specificantigen provides a highly effective “vaccine” that raises an immuneresponse directed against renal cell cancers. The chimeric molecule canbe used as a traditional vaccine or in adoptive immunotherapeuticapplications. Nucleic acids encoding a GM-CSF-G250 fusion protein can beused as naked DNA vaccines or to transfect cell in an adoptiveimmunotherapeutic treatment regimen.

Thus in one embodiment, this invention provides a construct comprising aG250 kidney cancer specific antigen attached to a granulocyte macrophagecolony stimulating factor (GM-CSF). The GM-CSF is preferably a humanGM-CSF, or a biologically active fragment and/or mutant thereof.Similarly the G250 antigen is a preferably a human G250 antigen. Inparticularly preferred embodiments the G250 antigen is covalentlyattached to the GM-CSF (directly or through a linker). Preferred linkersare encoded by the nucleotide sequence gcggcg. In a particularlypreferred embodiment the G250 antigen and the GM-CSF are components of afusion protein (chemically constructed or recombinantly expressed). Insuch fusion proteins, the G250 antigen and the GM-CSF are directlyjoined, or more preferably, joined by a peptide linker ranging in lengthfrom 2 to about 50, more preferably from about 2 to about 20, and mostpreferably from about 2 to about 10 amino acids. One preferred peptidelinker is -Arg-Arg-. A particularly preferred embodiment has thesequence of SEQ ID NO: 1 (excluding the His₆ (SEQ ID NO:26) tag).

In another embodiment this invention provides a composition comprisingthe chimeric molecules described herein and a pharmaceuticallyacceptable diluent or excipient.

This invention also provides a nucleic acid (e.g. a DNA or an RNA)encoding a fusion protein comprising a G250 kidney cancer specificantigen attached to a granulocyte macrophage colony stimulating factor(GM-CSF). The G250 is preferably a human G250 (or an antigenic fragmentor cancer-specific epitope thereof). Similarly the GM-CSF is apreferably a human GM-CSF or a biologically active fragment thereof. Inone preferred embodiment the nucleic acid encodes a fusion protein wherethe G250 antigen and the GM-CSF are directly joined, or more preferably,joined by a peptide linker ranging in length from 2 to about 50, morepreferably from about 2 to about 20, and most preferably from about 2 toabout 10 amino acids. In certain embodiments, the nucleic acid maypreferably encode a linker that is -Arg-Arg-. One preferred nucleic acidis the nucleic acid of SEQ ID NO: 2. In some preferred embodiments, thenucleic acid is a nucleic acid that encodes the polypeptide of SEQ IDNO: 1. The nucleic acid is preferably in an expression cassette and incertain embodiments, the nucleic acid is present in a vector (e.g. abaculoviral vector).

This invention also provides a host cell transfected with one or more ofthe nucleic acids described herein. The host cell is preferably aeukaryotic cell, and most preferably an insect cell.

This invention also provides methods of producing an anti-tumor vaccine.The methods preferably involve culturing a cell transfected with anucleic acid encoding a chimeric GM-CSF-G250 chimeric molecule underconditions where the nucleic acid expresses a G250-GM-CSF fusion proteinand recovering said fusion protein. Again the cell is preferably aeukaryotic cell, more preferably an insect (e.g. an SF9) cell.

In another embodiment, this invention provides methods of inducing animmune response against the G250 kidney-specific antigen, and/or a celldisplaying the G250 kidney-specific antigen, and/or any cancer cell thatexpresses a G250 antigen, and/or an antigen cross-reactive with a G250antigen. The methods involve activating a cell of the immune system witha construct comprising a kidney cancer specific antigen (G250) attachedto a granulocyte macrophage colony stimulating factor (GM-CSF) wherebythe activating provides an immune response directed against the G250antigen. In some embodiments, the activating comprises contacting anantigen presenting cell (e.g. monocyte, or dendritic cell) with theconstruct (chimeric molecule). In certain embodiments, the activatedcell is a cytotoxic T-lymphocyte (CTL), or a tumor infiltratinglymphocyte, etc. The activating can also involve contacting a peripheralblood lymphocyte (PBL) or a tumor infiltrating lymphocyte (TIL) with theconstruct. The contacting can take place in vivo, or ex vivo (e.g., invitro). In various embodiments, the activating comprises loading anantigen presenting cell (APC) with a polypeptide comprising a G250. Theactivation can also comprise transfecting a cell (e.g., a PBL, an APC, aTIL, a renal cell carcinoma tumor cell, etc.) with a nucleic acidencoding a GM-CSF-G250 fusion protein. The method may further compriseinfusing cells (e.g. cytotoxic T lymphocytes) back into the mammal.

In still another embodiment this invention provides a method ofinhibiting the proliferation or growth of a transformed (e.g.neoplastic) kidney cell. The method involves activating a cell of theimmune system with a construct comprising a kidney cancer specificantigen (G250) attached to a granulocyte macrophage colony stimulatingfactor (GM-CSF) whereby the activating provides an immune responsedirected against the G250 antigen and the immune response inhibits thegrowth or proliferation of a transformed kidney cell. In preferredembodiments, the transformed kidney cell is a renal cell carcinoma cell(e.g. in a solid tumor, a disperse tumor, or a metastatic cell). Theactivating can comprise contacting an antigen presenting cell (e.g. adendritic cell) with the construct. The activated cell can include, butis not limited to a cytotoxic T-lymphocyte (CTL) a tumor infiltratinglymphocyte (TIL), etc. In certain embodiments, the activating comprisesinjecting (or otherwise administering) to a mammal one or more of thefollowing: a polypeptide comprising a GM-CSF-G250 fusion protein;dendritic cells pulsed with a GM-CSF-G250 fusion protein; a gene therapyconstruct (e.g. adenovirus, gutless-adenovirus, retrovirus, lentivirus,adeno-associated virus, vaccinia virus, etc) comprising a nucleic acidencoding a GM-CSF-G250 fusion protein, a dendritic expressing aGM-CSF-G250 fusion protein, a tumor cell (e.g. RCC) expressing aGM-CSF-G250 fusion protein, a fibroblast expressing a GM-CSF-G250 fusionprotein, a GM-CSF-G250 naked DNA, a transfection reagent (e.g. cationiclipid, dendrimer, liposome, etc. containing or complexed with a nucleicacid encoding a GM-CSF-G250 polypeptide. In a particularly preferredembodiment, activating comprises activating isolated dendriticcells/PMBCs. In another embodiment, the activating comprises contacting(in vivo or ex vivo) a peripheral blood lymphocyte (PBL) or a tumorinfiltrating lymphocyte (TEL) with said construct. The peripheral bloodcells and/or dendritic cells and/or monocytes are preferably infusedinto the subject.

This invention also provides a method of inhibiting the proliferation orgrowth of a transformed renal cell that bears a G250 antigen. The methodinvolves removing an immune cell from a mammalian host; activating theimmune cell by contacting the cell with a protein comprising a renalcell carcinoma specific antigen (G250) attached to a granulocytemacrophage colony stimulating factor (GM-CSF) or a fragment thereof;optionally expanding the activated cell; and infusing the activated cellinto an organism containing a transformed renal cell bearing a G250antigen. In certain embodiments, the activating comprises contacting thecell with one or more of the following: a polypeptide comprising aGM-CSF-G250 fusion protein; dendritic cells pulsed with a GM-CSF-G250fusion protein; a gene therapy construct (e.g. adenovirus,gutless-adenovirus, retrovirus, lantivirus, adeno-associated virus,vaccinia virus, etc) comprising a nucleic acid encoding a GM-CSF-G250fusion protein, a dendritic expressing a GM-CSF-G250 fusion protein, atumor cell (e.g. RCC) expressing a GM-CSF-G250 fusion protein, afibroblast expressing a GM-CSF-G250 fusion protein, a GM-CSF-G250 nakedDNA, a transfection reagent (e.g. cationic lipid, dendrimer, liposome,etc. containing or complexed with a nucleic acid encoding a GM-CSF-G250polypeptide. In a particularly preferred embodiment, activatingcomprises activating isolated dendritic cells/PMBCs. In anotherembodiment, the activating comprises contacting (in vivo or ex vivo) aperipheral blood lymphocyte (PBL) or a tumor infiltrating lymphocyte(TIL) with said construct. The peripheral blood cells and/or dendriticcells and/or monocytes are preferably infused into the subject. Theremoving may comprise isolating and culturing peripheral bloodlymphocytes and/or monocytes, and/or dendritic cells from the mammalianhost. The infusing may involve infusing the cultured cells or activatedcells produced using the cultured cells into the host from which theimmune cell was removed.

In still another embodiment, this invention provides a method oftreating an individual having a renal cell cancer. The method involvessensitizing antigen presenting cells (e.g., PBMCs, dendritic cells,etc.) in vitro with a sensitizing-effective amount of a chimeric fusionprotein comprising a renal cell carcinoma specific antigen (G250)attached to a granulocyte macrophage colony stimulating factor (GM-CSF);and administering to an individual having said renal cell cancer ormetastasis a therapeutically effective amount of the sensitized antigenpresenting cells. In particularly preferred embodiments, the antigenpresenting cells are autologous to the individual or allogenic withmatched MHC. In certain embodiments, the sensitizing involves contactingperipheral blood lymphocytes or monocytes or dendritic cells withG250-GM-CSF fusion protein. In certain embodiments, the sensitizinginvolves contacting PBL, TIL, monocyte, dendritic cell with aG250-GM-CSF polypeptide and/or transfecting dendritic cell, APC, RCC,fibroblasts, with a nucleic acid encoding the chimeric fusion protein.

DEFINITIONS

The term “G250-GM-CSF” refers to a chimeric molecule comprising a G250renal cell tumor antigen attached to a granulocyte-macrophage colonystimulating factor. The attachment may be a chemical conjugation (director through a linker) or the chimeric molecule can be a fusion protein(recombinantly expressed or assembled by condensation of the two subjectmolecules). The notation “G250-GM-CSF” encompasses embodiments where theG250 and the GM-CSF are attached terminally or to an internal site andcontemplates attachment of the G250 molecule to either the amino orcarboxyl terminus of the GM-CSF. In addition, the term my encompasschimeric molecules comprising fragments or mutants of G250 where theG250 fragments retain the epitope recognized by antibodies thatspecifically target renal cell carcinomas bearing the G250 antigen.Similarly, the term my encompass chimeric molecules comprising fragmentsor mutants of GM-CSF where the GM-CSF retain the biological activity ofnative GM-CSF (e.g. are recognized by receptors that recognize nativeGM-CSF and/or show similar mitogenic activity, etc.)

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The term also includes variants on the traditional peptidelinkage joining the amino acids making up the polypeptide.

The terms “nucleic acid” or “oligonucleotide” or grammatical equivalentsherein refer to at least two nucleotides covalently linked together. Anucleic acid of the present invention is preferably single-stranded ordouble stranded and will generally contain phosphodiester bonds,although in some cases, as outlined below, nucleic acid analogs areincluded that may have alternate backbones, comprising, for example,phosphoramide (Beaucage et al. (1993) Tetrahedron 49(10):1925) andreferences therein; Letsinger (1970) J. Org. Chem. 35:3800; Sprinzl etal. (1977) Eur. J. Biochem. 81: 579; Letsinger et al. (1986) Nucl. AcidsRes. 14: 3487; Sawai et al. (1984) Chem. Lett. 805, Letsinger et al.(1988) J. Am. Chem. Soc. 110: 4470; and Pauwels et al. (1986) ChemicaScripta 26: 1419), phosphorothioate (Mag et al. (1991) Nucleic AcidsRes. 19: 1437; and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu etal. (1989) J. Am. Chem. Soc. 111: 2321, O-methylphosphoroamiditelinkages (see Eckstein, Oligonucleotides and Analogues: A PracticalApproach, Oxford University Press), and peptide nucleic acid backbonesand linkages (see Egholm (1992) J. Am. Chem. Soc. 114: 1895; Meier etal. (1992) Chem. Int. Ed. Engl. 31: 1008; Nielsen (1993) Nature, 365:566; Carlsson et al. (1996) Nature 380: 207). Other analog nucleic acidsinclude those with positive backbones (Denpcy et al. (1995) Proc. Natl.Acad. Sci. USA 92: 6097; non-ionic backbones (U.S. Pat. Nos. 5,386,023,5,637,684, 5,602,240, 5,216,141 and 4,469,863; Angew. (1991) Chem. Intl.Ed. English 30: 423; Letsinger et al. (1988) J. Am. Chem. Soc. 110:4470; Letsinger et al. (1994) Nucleoside & Nucleotide 13:1597; Chapters2 and 3, ASC Symposium Series 580, “Carbohydrate Modifications inAntisense Research”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al.(1994), Bioorganic & Medicinal Chem. Lett. 4: 395; Jeffs et al. (1994)J. Biomolecular NMR 34:17; Tetrahedron Lett. 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,Carbohydrate Modifications in Antisense Research, Ed. Y. S. Sanghui andP. Dan Cook. Nucleic acids containing one or more carbocyclic sugars arealso included within the definition of nucleic acids (see Jenkins et al.(1995), Chem. Soc. Rev. pp 169-176). Several nucleic acid analogs aredescribed in Rawls, C & E News Jun. 2, 1997 page 35. These modificationsof the ribose-phosphate backbone may be done to facilitate the additionof additional moieties such as labels, or to increase the stability andhalf-life of such molecules in physiological environments.

The term “immune cell” refers to a cell that is capable ofparticipating, directly or indirectly, in an immune response. Immunecells include, but are not limited to T-cells, B-cells, dendritic cells,cytotoxic T-cells, tumor infiltrating lymphocytes, etc.

As used herein, the term “activating” (e.g. as in activating a cell oractivating an immune response) includes direct activation as by contactwith the construct or by indirect activation as by contact with theconstruct or antigenic fragment via an antigen presenting cell (e.g. adendritic cell).

A “fusion protein” refers to a polypeptide formed by the joining of twoor more polypeptides through a peptide bond formed between the aminoterminus of one polypeptide and the carboxyl terminus of anotherpolypeptide. The fusion protein may be formed by the chemical couplingof the constituent polypeptides, or it may be expressed as a singlepolypeptide from nucleic acid sequence encoding the single contiguousfusion protein. A single chain fusion protein is a fusion protein havinga single contiguous polypeptide backbone.

A “spacer” or “linker” as used in reference to a fusion protein refersto a peptide that joins the proteins comprising a fusion protein.Generally a spacer has no specific biological activity other than tojoin the proteins or to preserve some minimum distance or other spatialrelationship between them. However, the constituent amino acids of aspacer may be selected to influence some property of the molecule suchas the folding, net charge, or hydrophobicity of the molecule.

A “spacer” or “linker” as used in reference to a chemically conjugatedchimeric molecule refers to any molecule that links/joins theconstituent molecules of the chemically conjugated chimeric molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a RT-PCR analysis of RCC tumor cells.

FIG. 2 illustrates FACS analysis of dendritic cells derived fromadherent PBMC cultures.

FIG. 3 illustrates upregulation of HLA antigen in dendritic cells byGM-CSF-G250 fusion protein.

FIG. 4 illustrates cytotoxicity of bulk PMBC modulated by G250-GM-CSFfusion protein (patient 1).

FIG. 5 illustrates cytotoxicity of bulk PBMC modulated by GM-cSF/G250fusion protein (patient 2).

FIGS. 6A, 6B and 6C illustrate the expression and purification ofGM-CSF-G250 fusion protein. FIG. 6A shows immunohistochemical stainingfor G250 and GM-CSF expression with anti-G250 and anti-GM-CSF antibodiesSf-9 cells infected with and without fusion gene recombinantbaculovirus. Magnification, ×100. FIG. 6B shows a Western blot analysisof 6×His-tagged GM-CSF-G250 fusion protein eluted from the Ni-NTAaffinity column using antiGM-CSF antibody (L=loading, BT=break through,W=wash). FIG. 6C shows a coomassive blue-stained SDS-PAGE of fusionprotein eluted from Ni-NTA affinity column (lane 1) and further purifiedwith SP Sepharose/FPLC (lane 2 and lane 3).

FIGS. 7A and 7B show a comparison of functional activity of recombinantGM-CSF and purified GM-CSF-G250 fusion protein. GM-CSF activity wasmeasured using the GM-CSF dependent human cell line, TF-1. The TF-1cells (2×104/well/ml) were cultured in the presence of serially dilutedamount of (FIG. 7A) recombinant GM-CSF or, (FIG. 7B) purifiedGM-CSF-G250 fusion protein as indicated. After a 5-day incubation thecultures were pulsed with 0.1 mCi tritiated thymidine for an additional12 h. The cultures were then harvested and the incorporated thymidinemeasured by scintillation counting.

FIGS. 8A, 8B, and 8C show the immunomodulatory effects of fusion proteinon dendrtic cells. FIG. 8A shows a double-color flow cytometric analysisof dendritic cells grown in GM-CSF (800 U/ml) plus IL-4 (1000 U/ml) orfusion protein (FP) plus IL-4. Cells were labeled with FITC and PEconjugated antibodies against cell surface markers of DC, as indicated.Cells that were larger than lymphocytes were selectively gated andnegative controls correspond to labeling with an isotype-matched controlantibody. This analysis is representative of 5 DC cultures. FIG. 8Bshows a flow cytometric analysis of HLA antigens of DC cultured inGM-CSF plus IL-4 or Fusion protein plus IL-4. Cells labeled with primaryantibody (HLA class I or class II) and FITC-conjugated secondaryantibody. This analysis is representative of four different DC derivedfrom four RCC patients. FIG. 8C shows a double-color flow cytometricanalysis of DC expressed CD83⁺CD19⁻ that cultured in the condition asindicated. Data, means of triplicate; bars, SD. This analysis isrepresentative of four different DC derived from four RCC patients.

FIG. 9 shows a time course of cytokine mRNA expression in PBMC that weretreated with GM CSF G250 fusion protein (FP) (2.7 mg/107 cells) forvarious time period as indicated and then harvested forsemi-quantitative RT-PCR analysis. The 32P-labeled PCR products wereseparated by electrophoresis through a 7% acrylamide gel. Gels weredried and subjected to autoradiography. Titrated standard was preparedfrom diluted RNA samples extracted from PBMC treated with FP for 24 h.

FIGS. 10A, 10B, 10C, and 10D show growth and cytotoxicity profiles ofpatient-derived PBMC stimulated with GM-CSF-G250 fusion protein. FIG.10A shows growth expansion of PBMC (patient #1) induced by variousimmunomodulatory strategies as indicated. Cell cultures were stimulatedwith FP on day 0, day 6, day 12 and day 18. Culture medium was changedweekly but maintained in a constant volume. Cell counts were performedon day 20. Expansion fold was calculated by division of final cellcounts per ml with cell counts per ml seeded on day 0 (3×105 cells/ml).Data, means of triplicate; bars, SD. This analysis is representative offour different PBMC cultures derived from four RCC patients, whichshowed a similar growth profile. FIG. 10B shows cytotoxicity of PBMC(patient #1) against autologous normal kidney cells, primary tumor cellsand lymph node derived tumor cells. Cytotoxicity was determined by 18-h51 Cr-release assay on day 21. Killing activity was expressed as thelytic units per 106 effector cells. Lytic units are defined as thenumber of effector cells capable of inducing 30% lysis. Spontaneousrelease for tumor target was <20% of maximal release. Data, means oftriplicate; bars, SD. FIG. 10C shows the inhibition of cytotoxicityagainst autologous LN tumor cells by antibodies specific to T cells andFLA antigens. Tumor target cells or PBMC were pretreated with respectiveantibody as indicated prior to cytotoxicity assay. Data, means oftriplicate; bars, SD. FIG. 10D: Semi-quantitative RT-PCR analysis ofG250 mRNA expression by normal kidney, primary tumor and LN derivedtumor derived from patient #1.

FIGS. 11A and 11B shows fusion protein induced G250 targeted and MHCrestricted T cell immunity. FIG. 11A shows cytotoxicity of PBMC againstautologous and allogenic tumor targets as indicated. PBMC cultures werepretreated with IL-4 (1000 U/ml) and FP (0.34 mg/ml) or IL-4 and GM-CSF(800 U/ml) for one week and then restimulated with IL-2 and FP or IL-2and GM-CSF weekly. Cytotoxicity was determined by 18-h 51Cr-releaseassay on day 35. Cytotoxicity against autologous tumor target wasmeasured in the presence of isotype control antibody or antibodiesspecific to HLA class I, HLA class II, CD3, CD4, or CD8. Data, means oftriplicate; bars, SD. FIG. 11B shows a phenotypic analysis of FPmodulated PBMC that expressed antitumor activity.

FIG. 12 shows a map of the vector pCEP4/GMCSF-G250 where the recombinantgene is inserted between KpnI and XhoI.

FIG. 13 digestion and electrophoresis of pCEP4/GMCSF-G250 Lane 1:pCEP4/GMCSF-G250. Lane 2: pCEP4/GMCSF-G250 digested with KpnI and XhoI.M: Molecular weight marker (1 kb PLUS DNA ladder (Gibco)

DETAILED DESCRIPTION

This invention provides a novel approach to the treatment (e.g.mitigation of symptoms) of a renal cell carcinoma or any type of cancerthat expresses G250 antigen (e.g. cervical cancer) or that expresses anantigen cross-reactive with G250. In particular this invention utilizesa chimeric molecule comprising a kidney cancer specific antigen (G250)attached to a granulocyte-macrophage colony stimulating factor (GM-CSF).Without being bound to a particular theory, it is believed that thischimeric molecule affords two modes of activity. Vaccination of patientswith advanced renal cell carcinoma using a chimeric G250-GM-CSF moleculewill result in activation of the patient's dendritic cells (DC), themost potent antigen presenting cells. The dendritic cells take upGM-CSF, e.g., via the GM-CSF receptor and the attached G250 antigen isco-transported by virtue of its attachment to the GM-CSF. The dendriticcells process the G250 antigen and present G250 peptide on HLA class Iwhich then activates G250 specific cytotoxic T cells (CD3⁺ CD8⁺) whichcan then lyse G250 positive kidney cancer cells. In addition, oralternatively, the G250 peptide is presented on HLA class II cells thatactivate G250 specific T helper cells which then activate or maintainthe killing activity of CTLs.

In certain embodiments, a nucleic acid encoding a G250-GM-CSF constructcan be administered as a “naked DNA” vaccine. In this approach, theorganism/patient is injected, e.g. intramuscularly, with a nucleic acidencoding a G250-GM-CSF fusion protein. The nucleic acid is expressedwithin the organism leading to the production of a G250-GM-CSF fusionprotein which then elicits an anti-renal cell carcinoma immune responseas described above.

In another embodiment, the chimeric G250-GM-CSF molecules can be used inadoptive immunotherapy. In this instance, the chimeric molecule (fusionprotein) or a nucleic acid encoding the chimeric molecule is used toactivate lymphocytes (e.g. T-cells) ex vivo. The activated lymphocytesare optionally expanded, ex vivo, and then re-infused back into thesubject (patient) where they specifically attack and lyse G250 positivetumor cells (e.g. kidney cells tumor or cervical cancer cells).

In particularly preferred embodiments, this invention utilizes one ormore of the following formulations:

-   -   1. A polypeptide comprising a GM-CSF-G250 fusion protein    -   2. Dendritic, or other cells, pulsed with a polypeptide        comprising a GM-CSF-G250 fusion protein;    -   3. GM-CSF-G250 encoding nucleic acids in a “gene therapy” vector        (e.g. adenovirus, gutless-adenovirus, retrovirus, lantivirus,        adeno-associated virus, vaccinia virus, etc.)    -   4. Dendritic cells transfected with a GM-CSF-G250-encoding        nucleic acid (e.g., via recombinant virus, plasmid DNA        transfection, and the like);    -   5. Tumor cells (e.g. RCC cells) comprising a nucleic acid        encoding a polypeptide comprising a GM-CSF-G250 fusion protein;    -   7. A nucleic acid encoding a GM-CSF-G250 (e.g. “naked DNA”); and    -   8. A nucleic acid encoding a polypeptide comprising a        GM-CSF-G250 complexed with a transfection agent (e.g.,        DMRIE/DOPE lipid, dendrimers, etc.)

Each of these formulations can be directly administered to an organism(e.g. a mammal having a cancer that expresses a G250 antigen or anantigen cross-reactive to a G250 antigen) or can be used in an adoptiveimmunotherapy context. In the latter approach, the adoptiveimmunotherapy preferably utilizes cells derived from peripheral blood(e.g. peripheral blood lymphocytes (PBLs) or cells derived from a tumor(e.g. tumor infiltrating lymphocytes (TILS)). Administration of theformulation results in activation and propagation of G250-targetedcytotoxic T cells in PBMC or TIL cultures. Infusion of the G250-targetedCTLs into the patient results in the development and maintenance of aG250-directed immune response.

The formulations identified above can also be administered directly to amammal for “in vivo” vaccination. Thus, for example, GM-CSF-G250polypeptides or nucleic acids endoding such polypeptides can beadministered to the organism as “traditional” vaccines. The otherimmunogenic formulations identified above, however, are also highlyactive in vivo and can also be “directly” administered to an organism asa “vaccine”. Thus, for example, dendritic cells pulsed with aGM-CSF-G250 fusion protein, dendritic, or other cells, transfected witha nucleic acid encoding a GM-CSF-G250 fusion protein, gene therapyvectors encoding a GM-CSF-G250 polypeptide, can all be administered toan organism where they induce and maintain a population of G250-directedcytotoxic T cells.

It was a discovery of this invention that the G250-GM-CSF chimericmolecules e.g. when used in vivo as a vaccine or in an adoptiveimmunotherapeutic modality induce a highly vigorous immune responsespecifically directed at renal cell carcinomas. The approach results inthe death or inhibition of neoplastic renal cells whether diffuse (e.g.motile metastatic cells) or aggregated (e.g. as in a solid tumor). Thesemethods can accompany administration of other agents (e.g.immunomodulatory or cytotoxic agents, such as cytokines or drugs).

It is recognized that the methods of this invention need not showcomplete tumor elimination (e.g. a “cure”) to be of value. Even a slightdecrease in the growth rate of a tumor, and/or in the propagation ofmetastatic, or other neoplastic, cells can be clinically relevantimproving the quality and/or duration of life. Of course, given the highefficacy observed, it is expected that the methods of this invention mayoffer a significant or complete degree of remission particularly whenused in combination with other treatment modalities (e.g. surgery,chemotherapy, interleukin therapy, TGFβ or IL-10 antisense therapy,etc.).

I. G250-GMCSF Chimeric Molecules and their Expression

This invention utilizes a chimeric molecule comprising a G250 kidneycancer-specific antigen attached to a granulocyte-macrophage colonystimulating factor (GM-CSF) to induce a cell-mediated immune responsetargeted to renal tumor cells. In a chimeric molecule, two or moremolecules that exist separately in their native state are joinedtogether to form a single molecule having the desired functionality ofall of its constituent molecules. In this instance, the constituentmolecules are the G250 antigen and GM-CSF respectively. The G250provides an epitope that is presented (e.g. to T-cells) resulting inactivation and expansion of those cells and the formation of cytotoxiccells (e.g. cytotoxic T lymphocytes, tumor infiltrating lymphocytes(TILs), etc.) that are direct to tumor cells bearing the G250 antigen.The GM-CSF acts both to stimulate components of the immune system (e.g.monocytes, dendritic cells, NK, PMN, PBMC, etc.) and to mediate uptakeof the associated G250 antigen by dendritic cells. In addition,particularly in adoptive immunotherapeutic modalities, the GM-CSF alsocan act as an adjuvant.

The attachment of the G250 antigen to the GM-CSF can be direct (e.g. acovalent bond) or indirect (e.g. through a linker). In addition, theG250 antigen and the GM-CSF proteins can be attached by chemicalmodification of the proteins or they can be expressed as a recombinantfusion protein. Detailed methods of producing the individual componentsand the chimeric molecule are provided below.

The G250 kidney tumor specific antigen is known to those of skill in theart (see, e.g., Oosterwijk et al. (1996) Molecular characterization ofthe Renal Cell Carcinoma-associated antigen G250, Proc. Natl. Acad.Sci., USA, 37: 461; Uemura et al., (1994) Internal Image Anti-IdiotypeAntibodies Related to Renal-Cell Carcinoma-Associated Antigen G250, Int.J. Cancer, 56: 609-614). The G250 nucleic acid sequence is publiclyavailable (see, e.g., GenBank Accession number X66839).

Nucleic acid sequence of G250 (SEQ ID NO: 23):gcccgtacac accgtgtgct gggacacccc acagtcagccgcatggctcc cctgtgcccc agcccctggc tccctctgttgatcccggcc cctgctccag gcctcactgt gcaactgctgctgtcactgc tgcttctgat gcctgtccat ccccagaggttgccccggat gcaggaggat tcccccttgg gaggaggctcttctggggaa gatgacccac tgggcgagga ggatctgcccagtgaagagg attcacccag agaggaggat ccacccggagaggaggatct acctggagag gaggatctac ctggagaggaggatctacct gaagttaagc ctaaatcaga agaagagggctccctgaagt tagaggatct acctactgtt gaggctcctggagatcctca agaaccccag aataatgccc acagggacaaagaaggggat gaccagagtc attggcgcta tggaggcgacccgccctggc cccgggtgtc cccagcctgc gcgggccgcttccagtcccc ggtggatatc cgcccccagc tcgccgccttctgcccggcc ctgcgccccc tggaactcct gggcttccagctcccgccgc tcccagaact gcgcctgcgc aacaatggccacagtgtgca actgaccctg cctcctgggc tagagatggctctgggtccc gggcgggagt accgggctct gcagctgcatctgcactggg gggctgcagg tcgtccgggc tcggagcacactgtggaagg ccaccgtttc cctgccgaga tccacgtggttcacctcagc accgcctttg ccagagttga cgaggccttggggcgcccgg gaggcctggc cgtgttggcc gcctttctggaggagggccc ggaagaaaac agtgcctatg agcagttgctgtctcgcttg gaagaaatcg ctgaggaagg ctcagagactcaggtcccag gactggacat atctgcactc ctgccctctgacttcagccg ctacttccaa tatgaggggt ctctgactacaccgccctgt gcccagggtg tcatctggac tgtgtttaaccagacagtga tgctgagtgc taagcagctc cacaccctctctgacaccct gtggggacct ggtgactctc ggctacagctgaacttccga gcgacgcagc ctttgaatgg gcgagtgattgaggcctcct tccctgctgg agtggacagc agtcctcgggctgctgagcc agtccagctg aattcctgcc tggctgctggtgacatccta gccctggttt ttggcctcct ttttgctgtcaccagcgtcg cgttccttgt gcagatgaga aggcagcacagaaggggaac caaagggggt gtgagctacc gcccagcagaggtagccgag actggagcct agaggctgga tcttggagaatgtgagaagc cagccagagg catctgaggg ggagccggtaactgtcctgt cctgctcatt atgccacttc cttttaactgccaagaaatt ttttaaaata aatatttata at

Similarly, the nucleic acid sequence of GM-CSF (e.g. human GM-CSF) iswell known to those of skill in the art (see, e.g., GenBank accessionno: E02287).

Nucleic acid sequence of GM-CSF (SEQ ID NO: 24):taaagttctc tggaggatgt ggctgcagag cctgctgctc ttgggcactg tggcctgcag catctctgca cccgcccgct cgcccagccc cagcacgcag ccctgggagc atgtgaatgc catccaggag gcccggcgtc tcctgaacct gagtagagacactgctgctg agatgaatga aacagtagaa gtcatctcagaaatgtttga cctccaggag ccgacctgcc tacagacccgcctggagctg tacaagcagg gcctgcgggg cagcctcaccaagctcaagg gccccttgac catgatggcc agccactacaagcagcactg ccctccaacc ccggaaactt cctgtgcaacccagattatc acctttgaaa gtttcaaaga gaacctgaag gactttctgc ttgtcatccc ctttgactgc tgggagccagtccaggagtg agaccggcca gatgaggctg gccaagccggggagctgctc tctcatgaaa caagagctag aaactcaggatggtcatctt ggagggacca aggggtgggc cacagccatggtgggagtgg cctggacctg ccctgggcac actgaccctgatacaggcat ggcagaagaa tgggaatatt ttatactgacagaaatcagt aatatttata tatttatatt tttaaaatatttatttattt atttatttaa gttcatattc catatttattcaagatgttt taccgtaata attattatta aaaatagctt cta

Using the known sequence information nucleic acids encoding G250,GM-CSF, or a chimeric G250-GM-CSF can be produced using standard methodswell known to those of skill in the art. For example, the nucleicacid(s) may be cloned, or amplified by in vitro methods, such as thepolymerase chain reaction (PCR), the ligase chain reaction (LCR), thetranscription-based amplification system (TAS), the self-sustainedsequence replication system (SSR), etc. A wide variety of cloning and invitro amplification methodologies are well known to persons of skill inthe art.

Examples of these techniques and instructions sufficient to directpersons of skill through many cloning exercises are found in Berger andKimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology 152Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al. (1989)Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3, Cold SpringHarbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook et al.);Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel); Cashionet al., U.S. Pat. No. 5,017,478; and Carr, European Patent No.0,246,864.

Examples of techniques sufficient to direct persons of skill through invitro amplification methods are found in Berger, Sambrook, and Ausubel,as well as Mullis et al., (1987) U.S. Pat. No. 4,683,202; PCR ProtocolsA Guide to Methods and Applications (Innis et al. eds) Academic PressInc. San Diego, Calif. (1990) (Innis); Arnheim & Levinson (Oct. 1, 1990)C&EN 36-47; The Journal Of NIH Research (1991) 3: 81-94; (Kwoh et al.(1989) Proc. Natl. Acad. Sci. USA 86: 1173; Guatelli et al. (1990) Proc.Natl. Acad. Sci. USA 87, 1874; Lomell et al. (1989) J. Clin. Chem., 35:1826; Landegren et al., (1988) Science, 241: 1077-1080; Van Brunt (1990)Biotechnology, 8: 291-294; Wu and Wallace, (1989) Gene, 4: 560; andBarringer et al. (1990) Gene, 89: 117.

In addition, the cloning and expression of a GM-CSF-G250 fusion gene isdescribed in Example 1. While the cloning and expression of arecombinant fusion protein is illustrated it will be appreciated thatthe G250 and GM-CSF proteins can be purchased and/or recombinantlyexpressed and then chemically coupled as described below.

The G250 and the GM-CSF molecules may be joined together in any order.Thus, the G250 can be joined to either the amino or carboxy termini ofthe GM-CSF. Where the molecules are chemically conjugated, they need notbe joined end to end and can be attached at any convenient terminal orinternal site.

The G250 and GM-CSF may be attached by any of a number of means wellknown to those of skill in the art. Typically the G250 and the GM-CSFare conjugated, either directly or through a linker (spacer). Becauseboth molecules are polypeptides, in one embodiment, it is preferable torecombinantly express the chimeric molecule as a single-chain fusionprotein that optionally contains a peptide spacer between the GM-CSF andthe G250.

Means of chemically conjugating molecules are well known to those ofskill. Polypeptides typically contain variety of functional groups;e.g., carboxylic acid (COOH) or free amine (—NH₂) groups, which areavailable for reaction with a suitable functional group on an effectormolecule to bind the effector thereto.

Alternatively, the G250 and/or the GM-CSF may be derivatized to exposeor attach additional reactive functional groups. The derivatization mayinvolve attachment of any of a number of linker molecules such as thoseavailable from Pierce Chemical Company, Rockford Ill.

A “linker”, as used herein, is a molecule that is used to join the G250to the GM-CSF. In preferred embodiments, the linker is capable offorming covalent bonds to both the G250 and GM-CSF. Suitable linkers arewell known to those of skill in the art and include, but are not limitedto, straight or branched-chain carbon linkers, heterocyclic carbonlinkers, or peptide linkers. In certain embodiments, the linkers may bejoined to amino acids comprising G250 and/or GM-CSF through their sidegroups (e.g., through a disulfide linkage to cysteine). However, in apreferred embodiment, the linkers will be joined to the alpha carbonamino and carboxyl groups of the terminal amino acids. The linker may bebifunctional, having one functional group reactive with a substituent onthe G250 and a different functional group reactive with a substituent onthe GM-CSF. Alternatively, the G250 and/or the GM-CSF may be derivatizedto react with a “mono-functional” linker (see, e.g., U.S. Pat. Nos.4,671,958 and 4,659,839 for procedures to generate reactive groups onpeptides).

In a particularly preferred embodiment, the chimeric molecules of thisinvention are fusion proteins. The fusion protein can be chemicallysynthesized using standard chemical peptide synthesis techniques, or,more preferably, recombinantly expressed. Where both molecules arerelatively short the chimeric molecule may be synthesized as a singlecontiguous polypeptide. Solid phase synthesis in which the C-terminalamino acid of the sequence is attached to an insoluble support followedby sequential addition of the remaining amino acids in the sequence is apreferred method for the chemical synthesis of the polypeptides of thisinvention. Techniques for solid phase synthesis are described by Baranyand Merrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in ThePeptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods inPeptide Synthesis, Part A., Merrifield, et al. J. Am. Chem. Soc., 85:2149-2156 (1963), and Stewart et al., Solid Phase Peptide Synthesis, 2nded. Pierce Chem. Co., Rockford, Ill. (1984).

In a most preferred embodiment, the chimeric fusion proteins of thepresent invention are synthesized using recombinant DNA methodology.Generally this involves creating a DNA sequence that encodes the fusionprotein, placing the DNA in an expression cassette under the control ofa particular promoter, expressing the protein in a host, isolating theexpressed protein and, if required, renaturing the protein.

DNA encoding the fusion protein of this invention (GM-CSF-G250) may beprepared by any suitable method, including, for example, cloning andrestriction of appropriate sequences or direct chemical synthesis bymethods such as the phosphotriester method of Narang et al. Meth.Enzymol. 68: 90-99 (1979); the phosphodiester method of Brown et al.,Meth. Enzymol. 68: 109-151 (1979); the diethylphosphoramidite method ofBeaucage et al., Tetra. Lett., 22: 1859-1862 (1981); and the solidsupport method of U.S. Pat. No. 4,458,066.

Chemical synthesis produces a single stranded oligonucleotide. This maybe converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill would recognize that whilechemical synthesis of DNA is limited to sequences of about 100 bases,longer sequences may be obtained by the ligation of shorter sequences.

Alternatively, subsequences may be cloned and the appropriatesubsequences cleaved using appropriate restriction enzymes. Thefragments may then be ligated to produce the desired DNA sequence.

In a preferred embodiment, DNA encoding fusion proteins of the presentinvention is using DNA amplification methods such as polymerase chainreaction (PCR). As illustrated in Examples 1 and 2. Thus, for example,GM-CSF is amplified using primers that introduce EcoRI and NotI sites(3′ and 5′ respectively), and G250 cDNA is amplified with primersintroducing NotI and His-stop-GbL II (5′ and 3′ respectively. Theamplification products are ligated (GM-CSF-NotI-G250-His-stop-Bgl II).

The constructs illustrated in Example 1 introduce a linker (gcggcg)between the nucleic acids encoding G250 and GM-CSF. The linker sequenceis used to separate GM-CSF and G250 by a distance sufficient to ensurethat, in a preferred embodiment, each domain properly folds into itssecondary and tertiary structures. Preferred peptide linker sequencesadopt a flexible extended conformation, do not exhibit a propensity fordeveloping an ordered secondary structure that could interact with thefunctional GM-CSF and G250 domains. Typical amino acids in flexibleprotein regions include Gly, Asn and Ser. Virtually any permutation ofamino acid sequences containing Gly, Asn and Ser would be expected tosatisfy the above criteria for a linker sequence. Other near neutralamino acids, such as Thr and Ala, also may be used in the linkersequence. Thus, amino acid sequences useful as linkers of GM-CSF andG250, in addition to the one illustrated in Example 1, include theGly₄SerGly₅Ser linker (SEQ ID NO:3) used in U.S. Pat. No. 5,108,910 or aseries of four (Ala Gly Ser) residues (SEQ ID NO:4), etc. Still otheramino acid sequences that may be used as linkers are disclosed inMaratea et al. (1985), Gene 40: 39-46; Murphy et al. (1986) Proc. Nat'l.Acad. Sci. USA 83: 8258-62; U.S. Pat. No. 4,935,233; and U.S. Pat. No.4,751,180.

The length of the peptide linker sequence may vary without significantlyaffecting the biological activity of the fusion protein. In onepreferred embodiment of the present invention, a peptide linker sequencelength of about 2 amino acids is used to provide a suitable separationof functional protein domains, although longer linker sequences also maybe used. The linker sequence may be from 1 to 50 amino acids in length.In the most preferred aspects of the present invention, the linkersequence is from about 1-20 amino acids in length. In the specificembodiments disclosed herein, the linker sequence is from about 2 toabout 15 amino acids, and is advantageously from about 2 to about 10amino acids. Peptide linker sequences not necessarily required in thefusion proteins of this invention.

Generally the spacer will have no specific biological activity otherthan to join the proteins or to preserve some minimum distance or otherspatial relationship between them. However, the constituent amino acidsof the spacer may be selected to influence some property of the moleculesuch as the folding, net charge, or hydrophobicity.

Where it is desired to recombinantly express either the G250, theGM-CSF, or the G250-GM-CSF fusion protein, the nucleic acid sequencesencoding the desired protein are typically operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements typically include a transcriptional promoter, an optionaloperator sequence to control transcription, a sequence encoding suitablemRNA ribosomal binding sites, and sequences that control the terminationof transcription and translation. The ability to replicate in a host,usually conferred by an origin of replication, and a selection gene tofacilitate recognition of transformants may additionally beincorporated.

The nucleic acid sequences encoding the fusion proteins may be expressedin a variety of host cells, including E. coli and other bacterial hosts,and eukaryotic host cells including but not limited to yeast, insectcells (e.g. SF9 cells) and various other eukaryotic cells such as theCOS, CHO and HeLa cells lines and myeloma cell lines. The recombinantprotein gene will be operably linked to appropriate expression controlsequences for each host. For E. coli this includes a promoter such asthe T7, trp, or lambda promoters, a ribosome binding site and preferablya transcription termination signal. For eukaryotic cells, the controlsequences will include a promoter and preferably an enhancer derivedfrom immunoglobulin genes, SV40, cytomegalovirus, etc., and apolyadenylation sequence, and may include splice donor and acceptorsequences. In one particularly preferred embodiment the GM-CSF-G250fusion gene is inserted into polyhedrin gene locus-based baculovirustransfer vector (e.g., pVL 1393, available from PharMingen) andexpressed in insect cells (e.g. SF9 cells).

The plasmids of the invention can be transferred into the chosen hostcell by well-known methods such as calcium chloride transformation forE. coli and calcium phosphate treatment or electroporation for mammaliancells. Cells transformed by the plasmids can be selected by resistanceto antibiotics conferred by genes contained on the plasmids, such as theamp, gpt, neo, and hyg genes.

Once expressed, the recombinant fusion proteins can be purifiedaccording to standard procedures of the art, including ammonium sulfateprecipitation, his tag capture, affinity columns, column chromatography,gel electrophoresis and the like (see, generally, R. Scopes, ProteinPurification, Springer-Verlag, N.Y. (1982), Deutscher, Methods inEnzymology Vol. 182: Guide to Protein Purification., Academic Press,Inc. N.Y. (1990)). Substantially pure compositions of at least about 90to 95% homogeneity are preferred, and 98 to 99% or more homogeneity aremost preferred for pharmaceutical uses. Once purified, partially or tohomogeneity as desired, the polypeptides may then be usedtherapeutically.

One of skill in the art would recognize that after chemical synthesis,biological expression, or purification, the G250, GM-CSF, or GM-CSF-G250protein may possess a conformation substantially different than thenative conformations of the polypeptide(s). In this case, it may benecessary to denature and reduce the polypeptide and then to cause thepolypeptide to re-fold into the preferred conformation. Methods ofreducing and denaturing proteins and inducing re-folding are well knownto those of skill in the art (See, Debinski et al. (1993) J. Biol.Chem., 268: 14065-14070; Kreitman and Pastan, (1993) Bioconjug. Chem.,4: 581-585; and Buchner, et al., (1992) Anal. Biochem., 205: 263-270).Debinski et al., for example, describe the denaturation and reduction ofinclusion body proteins in guanidine-DTE. The protein is then refoldedin a redox buffer containing oxidized glutathione and L-arginine.

One of skill would recognize that modifications can be made to theGM-CSF, G250, or GM-CSF-G250 proteins without diminishing theirbiological activity. Some modifications may be made to facilitate thecloning, expression, or incorporation of the constituent molecules intoa fusion protein. Such modifications are well known to those of skill inthe art and include, for example, a methionine added at the aminoterminus to provide an initiation site, or additional amino acids placedon either terminus to create conveniently located restriction sites ortermination codons. The recombinant expression of a GM-CSF-G250 fusionprotein is illustrated in Example 1.

II. In Vivo Protein Vaccination

Immunogenic compositions (e.g. vaccines) are preferably prepared fromthe G250-GM-CSF fusion proteins of this invention. The immunogeniccompositions including vaccines may be prepared as injectables, asliquid solutions, suspensions or emulsions. The active immunogenicingredient or ingredients may be mixed with pharmaceutically acceptableexcipients which are compatible therewith. Such excipients are wellknown to those of skill in the art and include, but are not limited towater, saline, dextrose, glycerol, ethanol, and combinations thereof.The immunogenic compositions and vaccines may further contain auxiliarysubstances, such as wetting or emulsifying agents, pH buffering agents,or adjuvants to enhance the effectiveness thereof.

The immunogenic G250-GM-CSF compositions may be administeredparenterally, by injection subcutaneous, intravenous, intradermal,intratumoral, or intramuscularly injection. Alternatively, theimmunogenic compositions formed according to the present invention, maybe formulated and delivered in a manner to evoke an immune response atmucosal surfaces. Thus, the immunogenic composition may be administeredto mucosal surfaces by, for example, the nasal or oral (intragastric)routes. Alternatively, other modes of administration includingsuppositories and oral formulations may be desirable. For suppositories,binders and carriers may include, for example, polyalkalene glycols ortriglycerides. Such suppositories may be formed from mixtures containingthe active immunogenic ingredient (s) in the range of about 0.5 to about10%, preferably about 1 to 2%. Oral formulations may include normallyemployed carriers such pharmaceutical grades of saccharine, celluloseand magnesium carbonate. These compositions can take the form ofsolutions, suspensions, tablets, pills, capsules, sustained releaseformulations or powders and contain about 1 to 95% of the activeingredient(s), preferably about 20 to about 75%.

The immunogenic preparations and vaccines are administered in a mannercompatible with the dosage formulation, and in such amount as will betherapeutically effective, immunogenic and protective. The quantity tobe administered depends on the subject to be treated, including, forexample, the capacity of the individual's immune system to synthesizeantibodies, and if needed, to produce a cell-mediated immune response.Precise amounts of active ingredient required to be administered dependon the judgment of the practitioner. However, suitable dosage ranges arereadily determinable by one skilled in the art and may be of the orderof micrograms to milligrams of the active ingredient(s) per vaccination.The antigenic preparations of this invention can be administered byeither single or multiple dosages of an effective amount. Effectiveamounts of the compositions of the invention can vary from 0.01-1,000μg/ml per dose, more preferably 0.1-500 μg/ml per dose, and mostpreferably 10-300 μg/ml per dose.

Suitable regimes for initial administration and booster doses are alsovariable, but may include an initial administration followed bysubsequent booster administrations. The dosage may also depend or theroute of administration and will vary according to the size of the host.

The concentration of the active ingredient (chimeric protein) in animmunogenic composition according to the invention is in general about 1to 95%.

Immunogenicity can be significantly improved if the antigens areco-administered with adjuvants. While the GM-CSF component of thechimeric molecule can, itself act as an adjuvant, other adjuvants can beused as well. Adjuvants enhance the immunogenicity of an antigen but arenot necessarily immunogenic themselves. Adjuvants may act by retainingthe antigen locally near the site of administration to produce a depoteffect facilitating a slow, sustained release of antigen to cells of theimmune system. Adjuvants can also attract cells of the immune system toan antigen depot and stimulate such cells to elicit immune responses.

Immunostimulatory agents or adjuvants have been used for many years toimprove the host immune responses to, for example, vaccines. Intrinsicadjuvants, such as lipopolysaccharides, normally are the components ofthe killed or attenuated bacteria used as vaccines. Extrinsic adjuvantsare immunomodulators which are formulated to enhance the host immuneresponses. Thus, adjuvants have been identified that enhance the immuneresponse to antigens delivered parenterally. Some of these adjuvants aretoxic, however, and can cause undesirable side-effects, making themunsuitable for use in humans and many animals. Indeed, only aluminumhydroxide and aluminum phosphate (collectively commonly referred to asalum) are routinely used as adjuvants in human and veterinary vaccines.The efficacy of alum in increasing antibody responses to diphtheria andtetanus toxoids is well established and a HBsAg vaccine has beenadjuvanted with alum

A wide range of extrinsic adjuvants can provoke potent immune responsesto antigens. These include saponins complexed to membrane proteinantigens (immune stimulating complexes), pluronic polymers with mineraloil, killed mycobacteria in mineral oil, Freund's incomplete adjuvant,bacterial products, such as muramyl dipeptide (MDP) andlipopolysaccharide (LPS), as well as lipid A, and liposomes.

To efficiently induce humoral immune responses (HIR) and cell-mediatedimmunity (CMI), immunogens are often emulsified in adjuvants. Manyadjuvants are toxic, inducing granulomas, acute and chronicinflammations (Freund's complete adjuvant, FCA), cytolysis (saponins andPluronic polymers) and pyrogenicity, arthritis and anterior uveitis (LPSand MDP). Although FCA is an excellent adjuvant and widely used inresearch, it is not licensed for use in human or veterinary vaccinesbecause of its toxicity.

III. In Vivo DNA Vaccination

In some preferred embodiments, nucleic acids encoding a G250-GM-CSFfusion protein are incorporated into DNA vaccines. The ability ofdirectly injected DNA, that encodes an antigenic protein, to elicit aprotective immune response has been demonstrated in numerousexperimental systems (see, e.g., Conry et al. (1994) Cancer Res., 54:1164-1168; Cox et al. (1993) Virol, 67: 5664-5667; Davis et al. (1993)Hum. Mole. Genet., 2: 1847-1851; Sedegah et al. (1994) Proc. Natl. Acad.Sci., USA, 91: 9866-9870; Montgomery et al. (1993) DNA Cell Bio., 12:777-783; Ulmer et al. (1993) Science, 259: 1745-1749; Wang et al. (1993)Proc. Natl. Acad. Sci., USA, 90: 4156-4160; Xiang et al. (1994)Virology, 199: 132-140, etc.).

Vaccination through directly injecting DNA, that encodes an antigenicprotein, to elicit a protective immune response often produces bothcell-mediated and humoral responses. Moreover, reproducible immuneresponses to DNA encoding various antigens have been reported in micethat last essentially for the lifetime of the animal (see, e.g.,Yankauckas et al. (1993) DNA Cell Biol., 12: 771-776).

As indicated above, DNA vaccines are known to those of skill in the art(see, also U.S. Pat. Nos. 5,589,466 and 5,593,971, PCT/US90/01515,PCT/US93/02338, PCT/US93/048131, PCT/US94/00899, and the priorityapplications cited therein. In addition to the delivery protocolsdescribed in those applications, alternative methods of delivering DNAare described in U.S. Pat. Nos. 4,945,050 and 5,036,006.

Using DNA vaccine technology, plasmid (or other vector) DNA thatincludes a sequence encoding a G250-GM-CSF fusion protein operablylinked to regulatory elements required for gene expression isadministered to individuals (e.g. human patients, non-human mammals,etc.). The cells of the individual take up the administered DNA and thecoding sequence is expressed. The antigen so produced becomes a targetagainst which an immune response is directed. In the present case, theimmune response directed against the antigen component of the chimericmolecule provides the prophylactic or therapeutic benefit to theindividual renal cell cancers.

The vaccines of this invention may be administered by a variety oftechniques including several different devices for administeringsubstances to tissue. The published literature includes several reviewarticles that describe aspects of DNA vaccine technology and cite someof the many reports of results obtained using the technology (see, e.g.,McDonnel and Askari (1996) New Engl. J. Med. 334(1): 42-45; Robinson(1995) Can. Med. Assoc. J. 152(10): 1629-1632; Fynan et al. (1995) Int.J. Immunopharmac. 17(2): 79-83; Pardoll and Beckerleg (1995) Immunity 3:165-169; and Spooner et al. (1995) Gene Therapy 2: 173-180.

According to the present invention, the G250-GM-CSF coding sequence isinserted into a plasmid (or other vector) which is then used in avaccine composition. In preferred embodiments, the G250-GM-CSF codingsequence is operably linked to regulatory elements required forexpression of the construct in eukaryotic cells. Regulatory elements forDNA expression include, but are not limited to a promoter and apolyadenylation signal. In addition, other elements, such as a Kozakregion, may also be included in the genetic construct. Initiation andtermination signals are regulatory elements which are often, but notnecessarily, considered part of the coding sequence. In preferredembodiments, the coding sequences of genetic constructs of thisinvention include functional initiation and termination signals.

Examples of promoters useful to practice the present invention,especially in the production of a genetic vaccine for humans, includebut are not limited to, promoters from Simian Virus 40 (SV40), MouseMammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV)such as the HIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV,Cytomegalovirus (CMV) such as the CMV immediate early promoter, EpsteinBarr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters fromhuman genes such as human Actin, human Myosin, human Hemoglobin, humanmuscle creatine and human metalothionein.

Examples of polyadenylation signals useful to practice the presentinvention, especially in the production of a genetic vaccine for humans,include but are not limited to SV40 polyadenylation signals and LTRpolyadenylation signals. In particular, the SV40 polyadenylation signalwhich is in pCEP4 plasmid (Invitrogen, San Diego, Calif.), referred toas the SV40 polyadenylation signal, may be used.

In addition to the regulatory elements required for DNA expression,other elements may also be included in the DNA molecule. Such additionalelements include enhancers. The enhancer may be selected from the groupincluding but not limited to, human Actin, human Myosin, humanHemoglobin, human muscle creatine and viral enhancers such as those fromCMV, RSV and EBV.

The present invention relates to methods of introducing genetic materialinto the cells of an individual in order to induce immune responsesagainst renal cell cancers. The methods comprise the steps ofadministering to the tissue of said individual, DNA that includes acoding sequence for a G250-GM-CSF fusion protein operably linked toregulatory elements required for expression. The DNA can be administeredin the presence of adjuvants or other substances that have thecapability of promoting DNA uptake or recruiting immune system cells tothe site of the inoculation. It should be understood that, in preferredembodiments, the DNA transcription unit itself is expressed in the hostcell by transcription factors provided by the host cell, or provided bya DNA transcriptional unit. A DNA transcription unit can comprisenucleic acids that encode proteins that serve to stimulate the immuneresponse such as a cytokine, proteins that serve as an adjuvant andproteins that act as a receptor.

Vectors containing the nucleic acid-based vaccine of the invention canbe introduced into the desired host by methods known in the art, e.g.,transfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, lipofection(lysosome fusion), use of a gene gun, or a DNA vector transporter (see,e.g., Wu et al (1992) J. Biol. Chem. 267: 963-967; Wu and Wu (1988) J.Biol. Chem. 263: 14621-14624). The subject can be inoculatedintramuscularly, intranasally, intraperatoneally, subcutaneously,intradermally, topically, or by a gene gun.

The subject can also be inoculated by a mucosal route. The DNAtranscription unit can be administered to a mucosal surface by a varietyof methods, including lavage, DNA-containing nose-drops, inhalants,suppositories or by microsphere encapsulated DNA. For example, the DNAtranscription unit can be administered to a respiratory mucosal surface,such as the trachea or into any surface including the tongue or mucousmembrane.

The DNA transcription units are preferably administered in a medium,i.e., an adjuvant, that acts to promote DNA uptake and expression.Preferably, a pharmaceutically acceptable, inert medium is suitable asan adjuvant for introducing the DNA transcription unit into the subject.One example of a suitable adjuvant is alum (alumina gel), though even asaline solution is acceptable. Other possible adjuvants include organicmolecules such as squalines, iscoms, organic oils and fats.

An immuno-effector can be co-expressed with the G250-GM-CSF nucleic acidof this present invention and thereby enhance the immune response to theantigen. A nucleic acid encoding the immuno-effector may be administeredin a separate DNA transcription unit, operatively linked to a suitableDNA promoter, or alternatively the immuno-effector may be included in aDNA transcription unit comprising a nucleic acid that encodes theG250-GM-CSF construct that are operatively linked to one or more DNApromoters. Other embodiments contain two or more such immuno-effectorsoperatively linked to one or more promoters. The nucleic acid canconsist of one contiguous polymer, encoding both the chimeric proteinand the immuno-effector or it can consist of independent nucleic acidsegments that individually encode the chimeric molecule and theimmuno-effector respectively. In the latter case, the nucleic acid maybe inserted into one vector or the independent nucleic acid segments canbe placed into separate vectors. The nucleic acid encoding theimmuno-effector and the chimeric molecule may be either operativelylinked to the same DNA promoter or operatively linked to separate DNApromoters. Adding such an immuno-effector is known in the art.Alternatively, soluble immuno-effector proteins (cytokines, monokines,interferons, etc.) can be directly administered into the subject inconjunction with the G250-GM-CSF DNA.

Examples of immuno-effectors include, but are not limited to,interferon-α, interferon-γ, interferon-β, interferon-θ, interferon-τ,tumor necrosis factor-α, tumor necrosis factor-β, interleukin-2,interleukin-6, interleukin-7, interleukin-12, interleukin-15, B7-1 Tcell co-stimulatory molecule, B7-2 T cell co-stimulatory molecule,immune cell adhesion molecule (ICAM)-1, T cell co-stimulatory molecule,granulocyte colony stimulatory factor, granulocyte-macrophage colonystimulatory factor, and combinations thereof.

When taken up by a cell, the genetic construct(s) may remain present inthe cell as a functioning extrachromosomal molecule and/or integrateinto the cell's chromosomal DNA. DNA may be introduced into cells whereit remains as separate genetic material, e.g., in the form of a plasmidor plasmids. Alternatively, linear DNA which can integrate into thechromosome may be introduced into the cell. When introducing DNA intothe cell, reagents which promote DNA integration into chromosomes may beadded. DNA sequences which are useful to promote integration may also beincluded in the DNA molecule. Alternatively, RNA may be administered tothe cell. It is also contemplated to provide the genetic construct as alinear minichromosome including a centromere, telomeres and an origin ofreplication. Gene constructs may remain part of the genetic material inattenuated live microorganisms or recombinant microbial vectors whichlive in cells. Gene constructs may be part of genomes of recombinantviral vaccines where the genetic material either integrates into thechromosome of the cell or remains extrachromosomal.

Genetic constructs can be provided with a mammalian origin ofreplication in order to maintain the construct extrachromosomally andproduce multiple copies of the construct in the cell. Thus, for example,plasmids pCEP4 and pREP4 from Invitrogen (San Diego, Calif.) contain theEpstein Barr virus origin of replication and nuclear antigen EBNA-1coding region which produces high copy episomal replication withoutintegration.

An additional element may be added which serves as a target for celldestruction if it is desirable to eliminate cells receiving the geneticconstruct for any reason. A herpes thymidine kinase (tk) gene in anexpressible form can be included in the genetic construct. The druggangcyclovir can be administered to the individual and that drug willcause the selective killing of any cell producing tk, thus, providingthe means for the selective destruction of cells with the G250-GM-CSFnucleic acid construct.

In order to maximize protein production, regulatory sequences may beselected which are well suited for gene expression in the cells intowhich the construct is administered. Moreover, codons may be selectedwhich are most efficiently transcribed in the cell. One having ordinaryskill in the art can produce DNA constructs which are functional in thecells.

The concentration of the dosage is preferably sufficient to provide aneffective immune response. The dosage of the recombinant vectorsadministered will depend upon the properties of the formulationemployed, e.g., its in vivo plasma half-life, the concentration of therecombinant vectors in the formulation, the administration route, thesite and rate of dosage, the clinical tolerance of the subject, and thelike, as is well within the skill of one skilled in the art. Differentdosages may be utilized in a series of inoculations; the practitionermay administer an initial inoculation and then boost with relativelysmaller doses of the recombinant vectors or other boosters.

The preferred dose range is between about 30 μg to about 1 mg DNA, andmore preferably between about 50 μg to 500 μg. Lower doses may be usedas plasmid expression and inoculation are optimized. Dosages may differfor adults in contrast to adolescents or children. The inoculation ispreferably followed by boosters.

IV. Adoptive Immunotherapy

Adoptive immunotherapy refers to a therapeutic approach for treatingcancer or infectious diseases in which immune cells are administered toa host with the aim that the cells mediate either directly or indirectlyspecific immunity to (i.e., mount an immune response directed against)tumor cells. In preferred embodiments, the immune response results ininhibition of tumor and/or metastatic cell growth and/or proliferationand most preferably results in neoplastic cell death and/or resorption.The immune cells can be derived from a different organism/host(exogenous immune cells) or can be cells obtained from the subjectorganism (autologous immune cells).

The immune cells are typically activated in vitro by a particularantigen (in this case G250), optionally expanded, and then re-infusedback into the source organism (e.g., patient). Methods of performingadoptive immunotherapy are well known to those of skill in the art (see,e.g., U.S. Pat. Nos. 5,081,029, 5,985,270, 5,830,464, 5,776,451,5,229,115, 690,915, and the like).

In preferred embodiments, this invention contemplates numerousmodalities of adoptive immunotherapy, e.g. as described above. In oneembodiment, dendritic cells (e.g. isolated from the patient orautologous dendritic cells) are pulsed with G250 or the G250-GM-CSFchimeric molecule and then injected back into the subject where theypresent and activate immune cells in vivo. In addition, oralternatively, the dentritic cells can be transfected with nucleic acidsencoding the G250-GM-CSF fusion protein and then re-introduced into apatient.

In another embodiment, modified macrophage or dendritic cell (antigenpresenting cells) are pulsed with G250-GM-CSF fusion proteins ortransfected with nucleic acids encoding a G250-GM-CSF fusion protein,and then used to stimulate peripheral blood lymphocytes or TIL inculture and activate G250-targeted CTLs that are then infused into thepatient.

Similarly, fibroblasts, and other APCs, or tumor cells (e.g. RCCs) aretransfected with a nucleic acid expressing a G250-GM-CSF and used toactivate tumor cells or PBLs ex vivo to produce G250 directed CTLs thatcan then be infused into a patient.

Similarly various “transfection agents” including, but not limited togene therapy vectors (e.g. adenovirus, gutless-adenovirus, retrovirus,lantivirus, adeno-associated virus, vaccinia virus etc), cationiclipids, liposomes, dendrimers, and the like, containing or complexedwith a nucleic acid encoding a G250-GM-CSF fusion protein areadministered to PBLs or to tumor cells (e.g. RCCs) ex vivo to produceG250 directed CTLs.

In one particularly preferred emobdiments, tumor cells (e.g. RCC cells)transfected to express a G250-GM-CSF protein are used to provide anoff-the-shelf vaccine effective against tumors expressing a G250 antigenor an antigen that is cross-reactive with G250.

Using the teachings provided herein, other therapeutic modalitiesutilizing G250-GM-CSF polypeptides or G250-GM-CSF nucleic acids can bereadily developed.

As indicated above, in one embodiment the immune cells are derived fromperipheral blood lymphocytes or TILs (e.g. derived from tumors/tumorsuspension). Lymphocytes used for in vitro activation include, but arenot limited to T lymphocytes, various antigen presenting cells (e.g.monocytes, dendritic cells, B cells, etc.) and the like. Activation caninvolve contacting an antigen presenting cell with the chimericmolecule(s) of this invention which then present the G250 antigen (orfragment thereof), e.g., on HLA class I molecules and/or on HLA class IImolecules, and/or can involve contacting a cell (e.g. T-lymphocyte)directly with the chimeric molecule. The antigen-presenting cells(APCs), including but not limited to macrophages, dendritic cells andB-cells, are preferably obtained by production in vitro from stem andprogenitor cells from human peripheral blood or bone marrow as describedby Inaba et al., (1992) J. Exp. Med. 176: 1693-1702.

Activation of immune cells can take a number of forms. These include,but are not limited to the direct addition of the chimeric molecule toperipheral blood lymphocytes (PBLs) or tumor infiltrating lymphocytes(TILs) in culture, loading of antigen presenting cells (e.g. monocytes,dendritic cells, etc.) with the chimeric molecule in culture,transfection of antigen presenting cells, or PBLs, with a nucleic acidencoding the GM-CSF-G250 chimeric fusion protein, and the like.

APC can be obtained by any of various methods known in the art. In apreferred aspect human macrophages and/or dendritic cells are used,obtained from human blood donors. By way of example but not limitation,PBLs (e.g. T-cells) can be obtained as follows:

Approximately 200 ml of heparinized venous blood is drawn byvenipuncture and PBL are isolated by Ficoll-hypaque gradientcentrifugation, yielding approximately 1 to 5×10⁸ PBL, depending uponthe lymphocyte count of the donor(s). The PBL are washed inphosphate-buffered saline and are suspended at approximately 2×10⁵/ml inRPMI 1640 medium containing 10% pooled heat-inactivated normal humanserum; this medium will be referred to as “complete medium.”

Similarly, other cells (e.g. mononuclear cells) are isolated fromperipheral blood of a patient (preferably the patient to be treated), byFicoll-Hypaque gradient centrifugation and are seeded on tissue culturedishes which are pre-coated with the patient's own serum or with otherAB+ human serum. The cells are incubated at 37° C. for 1 hr, thennon-adherent cells are removed by pipetting. To the adherent cells leftin the dish, is added cold (4° C.) 1 mM EDTA in phosphate-bufferedsaline and the dishes are left at room temperature for 15 minutes. Thecells are harvested, washed with RPMI buffer and suspended in RPMIbuffer. Increased numbers of macrophages may be obtained by incubatingat 37° C. with macrophage-colony stimulating factor (M-CSF); increasednumbers of dendritic cells may be obtained by incubating withgranulocyte-macrophage-colony stimulating factor (GM-CSF) as describedin detail by Inaba et al. (1992) J. Exp. Med. 176:1693-1702, and morepreferably by incubating with the G250-GM-CSF chimeric molecules of thisinvention and, optionally IL-4).

The cells (e.g. APCs) are sensitized by contacting/incubating them withthe chimeric molecule. In some embodiments, sensitization may beincreased by contacting the APCs with heat shock protein(s) (hsp)noncovalently bound to the chimeric molecule. It has been demonstratedthat hsps noncovalently bound to antigenic molecules can increase APCsensitization in adoptive immunotherapeutic applications (see, e.g.,U.S. Pat. No. 5,885,270).

In one preferred embodiment, e.g. as described in the examples herein,G250-GM-CSF fusion protein (with optional IL-4) is added into thepatients PBMC ex vivo and then cultured at 37° C. for 7 days. Theculture is re-stimulated weekly with IL-2 and fusion protein, e.g. for 4to 5 cycles until the culture shows anti-tumor activity againstautologous kidney tumor cells displaying G250. The CTLs are thenreinfused back into the patient.

For re-infusion, the cells are washed three times and resuspended in aphysiological medium preferably sterile, at a convenient concentration(e.g., 1×10⁷/ml) for injection in a patient. The cell suspension is thenfiltered, e.g., through sterile 110 mesh and put into Fenwall transferpacks. Samples of the cells are tested for the presence ofmicroorganisms including fungi, aerobic and anaerobic bacteria, andmycoplasma. A sample of the cells is optionally retained forimmunological testing in order to demonstrate induction of specificimmunity.

In a preferred embodiment, before use in immunotherapy, the stimulatedlymphocytes are tested for cell-mediated immune reactivity against tumorcells bearing the G250 antigen. The PBL/TIL, following stimulation withthe chimeric molecules of this invention can be examined with regard tocell surface expression of T and B cell markers by immunofluorescentanalysis using fluorescein-conjugated monoclonal antibodies to T and Bcell antigens. Expression of known T cell markers, such as the CD4 andCD8 antigens, confirms the identity of the activated lymphocytes as Tcells.

The activated cells (e.g. activated T cells) are then, optionally,tested for reactivity against G250. This could be accomplished by any ofseveral techniques known in the art for assaying specific cell-mediatedimmunity. For example, a cytotoxicity assay, which measures the abilityof the stimulated T cells to kill tumor cells bearing the G250 antigenin vitro, may be accomplished by incubating the lymphocytes withG250-bearing tumor cells containing a marker (e.g. ⁵¹Cr-labelled cells)and measuring ⁵¹Cr release upon lysis. Such assays have been described(see, e.g., Zarling et al. (1986) J. Immunol. 136: 4669). The activatedPBL could also be tested for T helper cell activity by measuring theirability to proliferate, as shown by ³H-thymidine incorporation,following stimulation, and/or by measuring their ability to producelymphokines such as IL-2 or interferon upon stimulation, in the absenceof exogenous IL-2. Other assays of specific cell-mediated immunity knownin the art, such as leukocyte-adherence inhibition assays (Thomson, D.M. P. (ed.), 1982, Assessment of Immune Status by the LeukocyteAdherence Inhibition Test, Academic Press, New York), may also be used.

Inoculation of the activated cells is preferably through systemicadministration. The cells can be administered intravenously through acentral venous catheter or into a large peripheral vein. Other methodsof administration (for example, direct infusion into an artery) arewithin the scope of the invention. Approximately 1×10⁸ cells are infusedinitially and the remainder are infused over the following severalhours. In some regimens, patients may optionally receive in addition asuitable dosage of a biological response modifier including but notlimited to the cytokines IFN-α, IFN-γ, IL-2, TNF or other cytokinegrowth factor, antisense TGFβ, antisense IL-10, and the like. Thus, insome patients, recombinant human IL-2 may be used and will be infusedintravenously every 8 hours beginning at the time of T cell infusion.Injections of IL-2 will preferably be at doses of 10,000 to 100,000units/kg bodyweight, as previously used in cancer patients (Rosenberg etal. (1985) N. Engl. J. Med. 313: 1485). The IL-2 infusion may becontinued for several days after infusion of the activated T cells iftolerated by the patient.

Treatment by inoculation of, e.g., activated T cells can be used aloneor in conjunction with other therapeutic regimens including but notlimited to administration of IL-2 (as described supra), otherchemotherapeutics (e.g. doxirubicin, vinblastine, vincristine, etc.),radiotherapy, surgery, and the like.

As indicated above, the cells may, optionally, be expanded in culture.This expansion can be accomplished by repeated stimulation of the Tcells with the G250-GM-CSF construct of this invention with or withoutIL-2 or by growth in medium containing IL-2 alone. Other methods of Tcell cultivation (for example with other lymphokines, growth factors, orother bioactive molecules) are also within the scope of the invention.For example, antibodies or their derivative molecules which recognizethe Tp67 or Tp44 antigens on T cells have been shown to augmentproliferation of activated T cells (Ledbetter et al. (1985) J. Immunol.135: 2331), and may be used during in vitro activation to increaseproliferation. Interferon has been found to augment the generation ofcytotoxic T cells (Zarling et al. (1978) Immunol. 121: 2002), and may beused during in vitro activation to augment the generation of cytotoxic Tcells against G250 bearing cancer cells.

The description provided above details various methods for isolation,activation, and expansion of PBL. However the present invention providesfor the use G250-GM-CSF constructs in various forms, and modificationsand adaptations to the method to accommodate these variations. Thusmodifications of various adoptive immunotherapeutic approaches utilizingthe G250-GM-CSF constructs are within the scope of the invention.

V. Gene Transfer for Systemic Therapy or for Adoptive Immunotherapy

In addition to use of the chimeric GM-CSF-G250 chimeric protein foractivation in adoptive immunotherapy, cells, (e.g., APCs, PBLs,fibroblasts, TILs, or RCC tumor cells) can be transfected with a vectorexpressing the chimeric molecule and used for adoptive immunotherapyand/or vaccine therapy.

In one preferred embodiment, the nucleic acid(s) encoding theGM-CSF-G250 chimeric fusion proteins are cloned into gene therapyvectors that are competent to transfect cells (such as human or othermammalian cells) in vitro and/or in vivo.

Several approaches for introducing nucleic acids into cells in vivo, exvivo and in vitro have been used. These include lipid or liposome basedgene delivery (WO 96/18372; WO 93/24640; Mannino and Gould-Fogerite(1988) BioTechniques 6(7): 682-691; Rose U.S. Pat. No. 5,279,833; WO91/06309; and Feigner et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7414) and replication-defective retroviral vectors harboring atherapeutic polynucleotide sequence as part of the retroviral genome(see, e.g., Miller et al. (1990) Mol. Cell. Biol. 10: 4239 (1990);Kolberg (1992) J. NIH Res. 4: 43, and Cornetta et al. (1991) Hum. GeneTher. 2: 215).

For a review of gene therapy procedures, see, e.g., Anderson, Science(1992) 256: 808-813; Nabel and Feigner (1993) TIBTECH 11: 211-217;Mitani and Caskey (1993) TIBTECH 11: 162-166; Mulligan (1993) Science,926-932; Dillon (1993) TIBTECH 11: 167-175; Miller (1992) Nature 357:455-460; Van Brunt (1988) Biotechnology 6(10): 1149-1154; Vigne (1995)Restorative Neurology and Neuroscience 8: 35-36; Kremer and Perricaudet(1995) British Medical Bulletin 51(1) 31-44; Haddada et al. (1995) inCurrent Topics in Microbiology and Immunology, Doerfler and Böhm (eds)Springer-Verlag, Heidelberg Germany; and Yu et al., (1994) Gene Therapy,1: 13-26.

Widely used retroviral vectors include those based upon murine leukemiavirus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immunodeficiencyvirus (SN), human immunodeficiency virus (HIV), alphavirus, andcombinations thereof (see, e.g., Buchscher et al. (1992) J. Virol. 66(5)2731-2739; Johann et al. (1992) J. Virol. 66 (5): 1635-1640 (1992);Sommerfelt et al., (1990) Virol. 176: 58-59; Wilson et al. (1989) J.Virol. 63: 2374-2378; Miller et al., J. Virol. 65: 2220-2224 (1991);Wong-Staal et al., PCT/US94/05700, and Rosenburg and Fauci (1993) inFundamental Immunology, Third Edition Paul (ed) Raven Press, Ltd., NewYork and the references therein, and Yu et al. (1994) Gene Therapy,supra; U.S. Pat. No. 6,008,535, and the like).

The vectors are optionally pseudotyped to extend the host range of thevector to cells which are not infected by the retrovirus correspondingto the vector. For example, the vesicular stomatitis virus envelopeglycoprotein (VSV-G) has been used to construct VSV-G-pseudotyped HIVvectors which can infect hematopoietic stem cells (Naldini et al. (1996)Science 272: 263, and Akkina et al. (1996) J Virol 70: 2581).

Adeno-associated virus (AAV)-based vectors are also used to transducecells with target nucleic acids, e.g., in the in vitro production ofnucleic acids and peptides, and in in vivo and ex vivo gene therapyprocedures. See, West et al. (1987) Virology 160: 38-47; Carter et al.(1989) U.S. Pat. No. 4,797,368; Carter et al. WO 93/24641 (1993); Kotin(1994) Human Gene Therapy 5: 793-801; Muzyczka (1994) J. Clin. Invst.94: 1351 for an overview of AAV vectors. Construction of recombinant AAVvectors are described in a number of publications, including Lebkowski,U.S. Pat. No. 5,173,414; Tratschin et al. (1985) Mol. Cell. Biol. 5(11):3251-3260; Tratschin, et al. (1984) Mol. Cell. Biol., 4: 2072-2081;Hermonat and Muzyczka (1984) Proc. Natl. Acad. Sci. USA, 81: 6466-6470;McLaughlin et al. (1988) and Samulski et al. (1989) J. Virol., 63:03822-3828. Cell lines that can be transformed by rAAV include thosedescribed in Lebkowski et al. (1988) Mol. Cell. Biol., 8: 3988-3996.Other suitable viral vectors include herpes virus, lentivirus, andvaccinia virus.

In addition to viral vectors, a number of non-viral transfection methodsare available. Such methods include, but are not limited toelectroporation methods, calcium phosphate transfection, liposomes,cationic lipid complexes, water-oil emulsions, polethylene imines, anddendrimers.

Liposomes were first described in 1965 as a model of cellular membranesand quickly were applied to the delivery of substances to cells.Liposomes entrap DNA by one of two mechanisms which has resulted intheir classification as either cationic liposomes or pH-sensitiveliposomes. Cationic liposomes are positively charged liposomes whichinteract with the negatively charged DNA molecules to form a stablecomplex. Cationic liposomes typically consist of a positively chargedlipid and a co-lipid. Commonly used co-lipids include dioleoylphosphatidylethanolamine (DOPE) or dioleoyl phosphatidylcholine (DOPC).Co-lipids, also called helper lipids, are in most cases required forstabilization of liposome complex. A variety of positively charged lipidformulations are commercially available and many other are underdevelopment. Two of the most frequently cited cationic lipids arelipofectamine and lipofectin. Lipofectin is a commercially availablecationic lipid first reported by Phil Feigner in 1987 to deliver genesto cells in culture. Lipofectin is a mixture of N-[1-(2,3-dioleyloyx)propyl]-N—N—N-trimethyl ammonia chloride (DOTMA) and DOPE.

DNA and lipofectin or lipofectamine interact spontaneously to formcomplexes that have a 100% loading efficiency. In other words,essentially all of the DNA is complexed with the lipid, provided enoughlipid is available. It is assumed that the negative charge of the DNAmolecule interacts with the positively charged groups of the DOTMA. Thelipid:DNA ratio and overall lipid concentrations used in forming thesecomplexes are extremely important for efficient gene transfer and varywith application. Lipofectin has been used to deliver linear DNA,plasmid DNA, and RNA to a variety of cells in culture. Shortly after itsintroduction, it was shown that lipofectin could be used to delivergenes in vivo. Following intravenous administration of lipofectin-DNAcomplexes, both the lung and liver showed marked affinity for uptake ofthese complexes and transgene expression. Injection of these complexesinto other tissues has had varying results and, for the most part, aremuch less efficient than lipofectin-mediated gene transfer into eitherthe lung or the liver.

PH-sensitive, or negatively-charged liposomes, entrap DNA rather thancomplex with it. Since both the DNA and the lipid are similarly charged,repulsion rather than complex formation occurs. Yet, some DNA doesmanage to get entrapped within the aqueous interior of these liposomes.In some cases, these liposomes are destabilized by low pH and hence theterm pH-sensitive. To date, cationic liposomes have been much moreefficient at gene delivery both in vivo and in vitro than pH-sensitiveliposomes. pH-sensitive liposomes have the potential to be much moreefficient at in vivo DNA delivery than their cationic counterparts andshould be able to do so with reduced toxicity and interference fromserum protein.

In another approach dendrimers complexed to the DNA have been used totransfect cells. Such dendrimers include, but are not limited to,“starburst” dendrimers and various dendrimer polycations.

Dendrimer polycations are three dimensional, highly ordered oligomericand/or polymeric compounds typically formed on a core molecule ordesignated initiator by reiterative reaction sequences adding theoligomers and/or polymers and providing an outer surface that ispositively changed. These dendrimers may be prepared as disclosed inPCT/US83/02052, and U.S. Pat. Nos. 4,507,466, 4,558,120, 4,568,737,4,587,329, 4,631,337, 4,694,064, 4,713,975, 4,737,550, 4,871,779,4,857,599.

Typically, the dendrimer polycations comprise a core molecule upon whichpolymers are added. The polymers may be oligomers or polymers whichcomprise terminal groups capable of acquiring a positive charge.Suitable core molecules comprise at least two reactive residues whichcan be utilized for the binding of the core molecule to the oligomersand/or polymers. Examples of the reactive residues are hydroxyl, ester,amino, imino, imido, halide, carboxyl, carboxyhalide maleimide,dithiopyridyl, and sulfhydryl, among others. Preferred core moleculesare ammonia, tris-(2-aminoethyl)amine, lysine, ornithine,pentaerythritol and ethylenediamine, among others. Combinations of theseresidues are also suitable as are other reactive residues.

Oligomers and polymers suitable for the preparation of the dendrimerpolycations of the invention are pharmaceutically-acceptable oligomersand/or polymers that are well accepted in the body. Examples of theseare polyamidoamines derived from the reaction of an alkyl ester of anα,β-ethylenically unsaturated carboxylic acid or an α,β-ethylenicallyunsaturated amide and an alkylene polyamine or a polyalkylene polyamine,among others. Preferred are methyl acrylate and ethylenediamine. Thepolymer is preferably covalently bound to the core molecule.

The terminal groups that may be attached to the oligomers and/orpolymers should be capable of acquiring a positive charge. Examples ofthese are azoles and primary, secondary, tertiary and quaternaryaliphatic and aromatic amines and azoles, which may be substituted withS or O, guanidinium, and combinations thereof. The terminal cationicgroups are preferably attached in a covalent manner to the oligomersand/or polymers. Preferred terminal cationic groups are amines andguanidinium. However, others may also be utilized. The terminal cationicgroups may be present in a proportion of about 10 to 100% of allterminal groups of the oligomer and/or polymer, and more preferablyabout 50 to 100%.

The dendrimer polycation may also comprise 0 to about 90% terminalreactive residues other than the cationic groups. Suitable terminalreactive residues other than the terminal cationic groups are hydroxyl,cyan, carboxyl, sulfhydryl, amide and thioether, among others, andcombinations thereof. However others may also be utilized.

The dendrimer polycation is generally and preferably non-covalentlyassociated with the polynucleotide. This permits an easy disassociationor disassembling of the composition once it is delivered into the cell.Typical dendrimer polycation suitable for use herein have a molecularweight ranging from about 2,000 to 1,000,000 Da, and more preferablyabout 5,000 to 500,000 Da. However, other molecule weights are alsosuitable. Preferred dendrimer polycations have a hydrodynamic radius ofabout 11 to 60 Å, and more preferably about 15 to 55 Å. Other sizes,however, are also suitable. Methods for the preparation and use ofdendrimers in gene therapy are well known to those of skill in the artand describe in detail, for example, in U.S. Pat. No. 5,661,025

Where appropriate, two or more types of vectors can be used together.For example, a plasmid vector may be used in conjunction with liposomes.In the case of non-viral vectors, nucleic acid may be incorporated intothe non-viral vectors by any suitable means known in the art. Forplasmids, this typically involves ligating the construct into a suitablerestriction site. For vectors such as liposomes, water-oil emulsions,polyethylene amines and dendrimers, the vector and construct may beassociated by mixing under suitable conditions known in the art.

VI. Administration of GM-CSF-G250 with Other Agents

In various embodiments, the GM-CSF-G250 fusion proteins, or nucleicacids encoding the GM-CSF-G250 fusion proteins can be administered inconjunction with other agents. Such agents include, but are not limitedto various chemotherapeutic agents (e.g. doxirubicin and derivatives,taxol and derivatives, vinblastine, vincristine, camptothecinderivatives, and the like, various cytokines (e.g. IL-2, IL-7, IL-12,IFN, etc.), various cytotoxins (e.g. Pseudomonas exotoxin andderivatives, diphtheria toxin and derivatives, ricin and derivatives,abrin and derivatives, thymidine kinase and derivatives), antisensemolecules (e.g. antisense IL-10, TGF-(β, etc.), antibodies againstvarious growth factors/receptors (e.g. anti-VEGF, anti-EGFR, anti-IL-8,anti-FGF etc.), and the like. The methods of this invention can also beused as a adjunct to surgery, and/or radiotherapy.

VII. Kits

Kits of the invention are provided that include materials/reagentsuseful for vaccination using a polypeptide antigen (GM-CSF-G250polypeptide) and/or DNA vaccination, and/or adoptive immunotherapy. Kitsoptimized for GM-CSF-G250 polypeptide vaccination preferably comprise acontainer containing a GM-CSF-G250 chimeric molecule. The molecule canbe provided in solution, in suspension, or as a (e.g. lyophilized)powder. The GM-CSF-G250 may be packaged with appropriatepharmaceutically acceptable excipient and/or adjuvant, e.g. in a unitdosage form.

Similarly, kits optimized for DNA vaccination of a construct encoding aGM-CSF-G250 polypeptide preferably comprise a container containing aGM-CSF-G250 nucleic acid (e.g. a DNA). As with the polypeptide, thenucleic acid can be provided in solution, in suspension, or as a (e.g.lyophilized) powder. The GM-CSF-G250 nucleic may be packaged withappropriate pharmaceutically acceptable excipient and/or facilitatingagent(s), e.g. in a unit dosage form. The kit can further includereagents and/or devices to facilitate delivery of the nucleic acid tothe subject (e.g. human or non-human mammal).

Kits optimized for adoptive immunotherapy typically include a containercontaining a chimeric GM-CSF-G250 polypeptide as described above. Thekits may optionally include a nucleic acid (e.g. a vector) encoding aGM-CSF-G250 fusion protein for ex vivo transfection of cells. Such kitsmay also, optionally, include various cell lines (e.g. RCC) and/orreagents (e.g. IL-2) to facilitate expansion of activated cells.

The kits can, optionally, include additional reagents (e.g. buffers,drugs, cytokines, cells/cell lines, cell culture media, etc.) and/ordevices (e.g. syringes, biolistic devices, etc.) for the practice of themethods of this invention.

In addition, the kits may include instructional materials containingdirections (i.e., protocols) for the practice of the methods of thisinvention. Thus typical instructional materials will teach the use ofGM-CSF-G250 chimeric molecules (or the nucleic acid encoding such) asvaccines, DNA vaccines, or adoptive immunotherapeutic agents in thetreatment of renal cell cancers. While the instructional materialstypically comprise written or printed materials they are not limited tosuch. Any medium capable of storing such instructions and communicatingthem to an end user is contemplated by this invention. Such mediainclude, but are not limited to electronic storage media (e.g., magneticdiscs, tapes, cartridges, chips), optical media (e.g., CD ROM), and thelike. Such media may include addresses to internet sites that providesuch instructional materials.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Cloning and Expression of GM-C SF and G250 Fusion Protein

This example describes the cloning, expression and purification of aGM-CSF-G250 fusion protein.

Cloning of Human GM-CSF-G250 Fusion Gene

A full-length human GM-CSF cDNA (0.8 kb) was cleaved from plasmidp91023(B) vector (Wong et al. (1985) Science 228: 810-815) with Eco RIrestriction endonuclease. Eco RI (5′) and Not I sites (3′) were insertedinto the GM-CSF cDNA by PCR (0.4 kb) using a first primer:

gcgggaattc(atg)tggctgcagagc (5′ GM-CSF Eco RI underlined, SEQ ID NO: 5)and a second primer: gagggaggcggccgc(ctc)ctggactggctc (3′GM-CSF Not I underlined, to remove stop codon, SEQ ID NO: 6).

NotI (5′) and His-Stop-Gbl II (3′) sites were introduced into fulllength G250 cDNA (1.6 kb) by PCR using the following primers:

5′ G250 (NotI): gagggagcggcc(gct)cccctgtgcccc(remove start codon, SEQ ID NO: 7), 3′ G250-His-stop codon- Bgl II:(1) gcagaggtagagatct(cta)atggtgatggtgatggtgg gctccagtctcggctacctc (SEQ ID NO: 8; brackets = stop last, second  underline =8 His (SEQ ID NO: 27)),  (2) ggagagatct(cta)atgatgatgatgatgatgatgatgggctccagtctcggctacctct  (SEQ ID NO: 9, brackets-stop last, secondunderline = 8 His (SEQ ID NO: 27)).

Fragments were ligated producing M-CSF-NotI-G250-His-stop-Bgl II asfollows:

5′ (gag)ggcggcc(gct)cccctgtgccccc (rest of Gm-CSF-G250) (SEQ ID NO:10;where (gag) is the last of GM-CSF, and (gct) is the first of G250.

The 250 fusion gene was inserted into the polyhedrin gene locus-basebaculovirus transfer vector pVL 1393 (PharMingen). In particular, theplasmid pVL 1393 was cut with Eco RI and Bgl II restrictionendonucleases and the Eco RI-GM-CSF-G250-his-stop-Bgl II construct wasinserted into the cut vector.

Insect cells (sf8 cells) were transfected using the BaculoGoldtransfection kit (Pharmingen). This involved co-transfection oflinearized BaculoGold virus DNA and recombinant plasmid DNA containingGM-CSF-G250 fusion gene into insect cells (sf8 cells). The recombinantbaculoviruses were amplified and the plaques were assayed to titer thevirus.

The G250-GM-CSF protein was purified according to protocols provided inthe Pharmigen Instruction Manual, 4th Edition, July 1997, page 41.Briefly beads were prepared by resuspending the Ni-NTA agarose beads.Two ml of beads were poured into 10 ml chromatography column (bindingapproximately 7.5-15 mg of 6×His fusion protein). The beads were allowedto settle in the column and the ethanol preservative was drained. Thebeads were then washed with 6×His wash buffer (Cat #21472A, Pharmigen)with 7.5 ml of 6×His wash buffer twice.

A cell lysate preparation was prepared by resuspending a cell pellet inice-cold insect cell lysis buffer (Cat #21425A, Pharmigen) containingreconstituted protease inhibitor cocktail (Cat#21426Z, Pharmigen). Thecells were lysed on ice for 45 minutes using 1 ml of lysis buffer per2×10⁷ cells). The lysate was transferred into a clean centrifuge tubeand centrifuged at 10,000 rpm for 30 minutes or filtered through a 0.22μm filter. The supernatant was saved for the column and the pellet wasdiscarded. 150 μl lysate was saved for an SDS-PAGE gel or Western blotand protein concentration determination.

Lysate was added to equilibrated Ni-NTA agarose beads for affinitypurification on a column. The supernatant was loaded slowly or the beadswere bound in a 15 ml conical tube with the lysate for 1 hour at 4° C.The flow-through fraction was saved.

The column was washed with 10-15 ml of 6×His wash buffer (Cat #21472A,Pharmigen) and the column was allowed to drain without drying. The washstep was repeated until the wash A₂₈₀ was less than 0.01 (approximately4 washes).

The fusion protein was then eluted with imidazole. Briefly 4.5 ml of the6×His elution buffer (Cat #21476A, Pharmigen) including imidazole wasadded as follows:

-   -   i. 0.1 M imidazole with 6×His elution buffer;    -   ii 0.2 M imidazole with 6×His elution buffer;    -   iii 0.3 M imidazole with 6×His elution buffer;    -   iv 0.4 M imidazole with 6×His elution buffer;    -   v 0.5 M imidazole with 6×His elution buffer;        The elution speed was maintained at a rate less than or equal to        1 ml per minute. The eluted fractions were collected (200 μL).

After analysis with SDS-PAGE gel and Western Blot, the clean and correctfractions were picked and pooled, dialyzed against PBS and the Ni-NTApurification repeated again.

Once more after analysis with SDS-PAGE gel and Western Blot, the cleanand correct fractions were picked and pooled, dialyzed against PBS.Further purification was performed on a Q-sepharose column. 1.0 ml ofthe column was equilibrated with 50 mM NaCl Buffer X (20 mM Hepes, 1 mMEDTA, 20% Glycerol, and 0.5 mM PMSF). The protein sample was loaded ontothe column and the flow-through fraction was collected.

Further purification was performed on a SP-sepharose column. 1.0 ml ofSP-sepharose column was loaded with 50 mM NaCl BufferX (20 mM Hepes, 1mM EDTA, 20% Glycerol, and 0.5 mM PMSF). The protein sample was loadedonto the column and the flow-through fraction was collected and saved.The column was eluted with the gradient salt buffer (50 mM NaCl BufferX-1000 mM fraction are 5.0 ml and 0.1 ml, respectively). Elution waswith 1000 mM NaCl buffer X.

The correct fractions were pooled and dialyzed against PBS.

FIG. 1 illustrates a RT-PCR analysis of RCC tumor cells. FIG. 2illustrates FACS analysis of dendritic cells derived from adherent PBMCcultures. FIG. 3 illustrates upregulation of HLA antigen in dendriticcells by GM-CSF-G250 fusion protein. FIG. 4 illustrates cytotoxicity ofbulk PMBC modulated by G250-GM-CSF fusion protein (patient 1).

Table 1 shows phenotypic modulation of bulk peripheral blood monocytesby GM-CSF-G250 fusion protein, IL-4, and IL-2 (patient #1).

Phenotype Day 7 Day 21 CD3⁺CD56⁻ 60 94 CD3⁻CD56⁺ 10 3 CD3⁺CD8⁺ 21 25CD3⁺CD4⁺ 40 68 CD3⁺TcR⁺ 54 90 CD3⁺CD25⁺ 10 25Table 2 shows phenotypic modulation of bulk peripheral blood monocytesby GM-CSF-G250 fusion protein, IL-4, and IL-2 for patient #2.

Phenotype Day 7 Day 21 Day 42 CD3⁺CD56⁻ 70 90 100 CD3⁻CD56⁺ 13 1 0CD3⁺CD8⁺ 21 22 17 CD3⁺CD4⁺ 48 74 86 CD3⁺TcR⁺ 62 91 97 CD3⁺CD25⁺ 10 28 16

Example 2 Induction of G250 Targeted and T-Cell Mediated Anti-TumorActivity Against Renal Cell Carcinoma Using a Chimeric Fusion ProteinConsisting of G250 and Granulocyte-Monocyte Colony Stimulating Factor

Immunotherapy targeting the induction of a T-cell mediated anti-tumorresponse in patients with renal cell carcinoma (RCC) holds significantpromise. Here we describe a new RCC vaccine strategy that allows for theconcomitant delivery of dual immune activators: G250, a widely expressedRCC associated antigen, and granulocyte-macrophage colony-stimulatingfactor (GM-CSF), an immunomodulatory factor for antigen presenting cells(APC). The G250-GM-CSF fusion gene was constructed and expressed in SF-9cells using a baculovirus expression vector system. The 66 kDa fusionprotein (FP) was subsequently purified through a 6×His-Ni-NTA affinitycolumn and a SP Sepharose/FPLC. The purified FP possessed GM-CSFbioactivity, comparable to that of recombinant GM-CSF when tested in aGM-CSF dependent cell line. When combined with IL-4 (1000 U/ml), FP(0.34 mg/ml) induced differentiation of monocytes (CD14⁺) into dendriticcells (DC) that express surface marker characteristic for APC.Up-regulation of mature DC (CD83+CD19−) (17% vs 6%) with enhanced HLAclass I and class II antigen expression was detected in FP cultured DCas compared to DC cultured with recombinant GM-CSF. Treatment of PBMCwith FP alone (2.7 mg/107 cells) augmented both Th₁ and Th₂ cytokinemRNA expression (IL-2, IL-4, GM-CSF, IFN-γ and TNF-α). When compared tovarious immune manipulation strategies in the long-term cultures of bulkPBMC, cells treated with FP (0.34 mg/ml) plus IL-4 (1000 U/ml) for oneweek and then re-stimulated with FP weekly plus IL-2 (201 U/ml) inducedthe most growth expansion of T cells expressing T cell receptor (TcR).Moreover, under such immunomodulatory manipulation, RCC specificcytotoxicity that could be blocked by anti HLA class I, anti-CD3 andanti-CD8 antibodies was demonstrated in four out of six tested PBMCcultures. In one tested patient, an augmented cytotoxicity against lymphnode (LN) derived RCC target was determined as compared to that againstprimary tumor targets, which corresponded to an eight-fold higher G250expression in LN tumor as compared to primary tumor. The replacement ofFP with recombinant GM-CSF completely abrogated the selection of RCCspecific killer cells. All FP modulated PBMC cultures with antitumoractivity showed an up-regulated CD3⁺ CD4⁺ cell population. These resultsindicate that GM-CSF-G250 FP is a potent immunostimulant with thecapacity for activating immunomodulatory DC and inducing a T-helper cellsupported, G250 targeted, and CD8′ mediated anti-tumor response. Thesefindings have important implications for the use of GM-CSF-G250 FP as atumor vaccine for the treatment of patients with advanced kidney cancer.

Introduction

Metastatic renal cell carcinoma (mRCC) poses a therapeutic challengebecause of its resistance to conventional modes of therapy such aschemotherapy and radiation therapy (Figlin (1999) J. Urol. 61: 381-387).Advances in the treatment of mRCC have evolved significantly in the lastdecade since the FDA approval of interleukin-2 (IL-2) in 1992. It hasbecome clear that immunotherapy is capable of producing durableremissions in selected RCC patients, yet the overall response rates ofimmunotherapy remain approximately 25% at best (Fisher et al. (1997)Cancer J. Sci. Amer., 3: S70) at the cost of measurable toxicities tothe patient. The recent identification of MHC restrictedtumor-associated antigens (TAA) and the understanding of the criticalrole of immunomodulatory dendritic cells (DC) have provided therationale for the development of tumor vaccines for cancer therapy (Wanget al. (1999) J. Mol. Med., 77: 640-655; Xu et al. (2000) Trends inBiotech. 18: 167-172). Many cancer vaccine strategies have been designedand tested in both animal models and human trials with encouragingresults. These include peptide-based vaccines (Rosenberg et al. (1999)J. Immunol., 163: 1690-1695; Parkhurst et al. (1996) J. Immunol., 157:2539-2548), dendritic cell (DC)-based vaccines (Yang et al. (2000) J.Immunol., 164: 4204-4211; Condon et al. (1996) Nature Med., 2:1122-1128; Zhou et al. (1996) Human Gene Ther., 10: 2719-2724; Nestle etal. (1998) Nature Med., 4: 328-332; Mulders et al. (1999) Clin. CancerRes., 5: 445-454), recombinant viruses/DNA/RNA based vaccines (Ulmer etal. (1998) J. Virol., 72: 5648-5653; Ying et al. (1999) Nature Med., 5:823-827; Liu (1998) Nature Med., 4: 515-519), and gene modified tumorcells (Mach et al. (1999) Cancer Res., 60: 3239-3246). Despite that thefact that RCC is thought to be a relatively immunogenic tumor, no RCCassociated antigens have been identified and characterized inassociation with a significant rationale for the development of a kidneycancer targeted tumor vaccine (Gaugler et al. (1996) Immunogenetics, 44:323-330; Brändle et al. (1996) J. Exp. Med., 183: 2501-2508; Brossart etal. (1998) Cancer Res., 58: 732-736).

The first widely expressed RCC tumor associated antigen that containsHLA-A2 restricted CTL epitopes has been recently identified and clonedfrom a RCC cell line (Grabmaier et al. (2000) Intl. J. Cancer, 85:865-870; Vissers et al. (1999) Cancer Res., 59: 5554-5559). This RCCassociated transmembrane protein, designated as G250, has been proven tobe identical to MN/CAIX, a TAA expressed in cervical cancer (Opayský etal. (1996) Genomics, 33: 480-487). Immunohistochemical staining withmAbG250 revealed that more than 75% of primary and metastatic RCCexpressed G250 while little to no expression was detected in the normalkidney (Grabmaier et al. (2000) Intl. J. Cancer, 85: 865-870). Inaddition, G250 expression is found in nearly all clear cell cancers ofthe kidney, the most common RCC variant, which provides further basisfor the use of G250 as a significant immune target for anti-cancertherapy. Antigen presentation is a crucial first step for vaccine-basedimmunotherapy. We therefore hypothesized that a chimeric proteinconsisting of G250 and GM-CSF, an immunomodulatory factor for thegeneration of functional DC, would augment vaccine capacity as comparedto the use of either agent alone. Several chimeric fusion proteinscontaining GM-CSF have been reported and have shown a variety of complexbiological effects dependent on their fusion components (Hall et al.(1999) Leukemia, 13: 629-633; Tripathi et al. (1999) Hybridoma 18:193-202; Battaglia et al. (2000) Exp. Hematol., 28: 490-498; Batova etal. (1999) Clin. Cancer Res., 5: 4259-4263). GM-CSF has been wellcharacterized as a growth factor that induces the proliferation andmaturation of myeloid progenitor cells (Hill et al. (1995) J. LeukocyteBiol. 58: 634-642). It enhances macrophage and granulocyte naturalcytotoxicity against tumor cells (Parhar et al. (1992) Europ. CytokineNetwork, 3: 299-306). The function of GM-CSF as a key factor for thedifferentiation of DC further substantiates its adjutant impact inimmune based vaccine therapy (Jonuleit et al. (1996) Archives ofDermatological Res. 289: 1-8). Direct evidence of the adjuvant effectsof GM-CSF in vaccine based immunotherapy has been demonstrated in animalmodels. Immunization with tumor peptide at skin sites containingepidermal DC newly recruited by pre-treatment with DNA encoding GM-CSFelicited an antigen specific T cell response, whereas peptideimmunization of control skin site showed no immune response (Bowne etal. (1999) Cytokines Cellu. Mol. Ther., 5: 217-225). Likewise, treatmentof established tumor with a hybridized cellular vaccine generated byfusing GM-CSF gene-modified DC with melanoma cells showed a greatertherapeutic efficacy when compared to the treatment with hybridizedvaccine generated with non-modified DC (Cao et al. (1999) Immunol., 97:616-625). An initial Phase I trial further demonstrated that systemicinjection of GM-CSF and IL-4 was capable of inducing tumor regressionand stable disease response in patients with advanced RCC and prostatecancer (Roth et al. (2000) Cancer Res., 60: 1934-1941). Similarly,vaccination of patients with irradiated autologous RCC or melanoma cellsengineered to secrete human GM-CSF also induced a potent anti-tumorimmunity (32 Simons et al. (1997) Cancer Res. 57: 1537-1546; Soiffer etal. (1998) Proc. Natl. Acad. Sci., USA, 5: 13141-13146).

In this example, we describe a strategy to generate fusion proteins (FP)consisting of G250 and GM-CSF. In addition, we tested the feasibility ofusing this non-viral and non-cellular RCC tumor vaccine as animmunostimulant for the in vitro modulation of DC and induction of G250targeted anti tumor response in PBMC cultures, that were derived frompatients with advanced kidney cancer.

Materials and Methods

Cloning of GM-CSF-G250 Fusion Gene in pVL 1393 Vector

Plasmid p91023(B)-GM-CSF (Wong et al. (1985) Science 228: 810-815) wasdigested with EcoRI, and the 0.8 kb fragment containing the full lengthof GM-CSF cDNA was used to generate the 0.4 kb GM-CSF fragmentcontaining the functional epitope flanked by an EcoR I site on the 5′side and a Not I site on the 3′ side replacing the GM-CSF stop codon, byDNA PCR. The GM-CSF fragment from the PCR product was subcloned into theEcoR I and Bgl II sites of polyhedrin gene locus-based baculovirustransfer vector pVL1393 (Pharmigen, San Diego, Calif.). Similarly,pBM20CMVG250 Osterwick (2000) Int. J. Cancer, 85: A65-A70) was used toamplify the full length of G250 cDNA (1.6 kb) containing a Not I sitefollowed by a 6 nucleotide linker coding for 2 arginines by PCR in the5′-flanking region of the G250 after the removal of its start codon. The3′-flanking region of G250 was designed to encode 6 histidines followedby the stop codon and Bgl II site. The G250 fragment was gel purified.Both the vector pVL1393 already contained the GM-CSF and the G250 PCRamplified fragments were cut out with Not I and Bgl II. The G250fragment and the vector were ligated for 3 hr at 16° C., and latertransformed and plated on LB plates. The colonies containing the correctplasmid were purified by cesium chloride buoyant ultracentrifugation.The plasmid was cut with a set of different restriction enzymes toverify the plasmids. The plasmid clones were further verified forhistidine tag using an Amplicycle Sequencing Kit (Perkin Elmer).

Generation and Purification of Fusion Protein

The recombinant baculovirus containing His-tagged GM-CSF-G250 fusiongene was generated by co-transfection of 0.5 mg BaculoGold DNA (modifiedAcNPV baculovirus DNA) (Pharmigen, San Diego, Calif.) and 5 mg ofpVL1393/GM-CSF-G250 in Sf9 cells (Spodoptera frugiperda). Viruses werefurther amplified at a low MOI (<1) in adherent Sf9 insect cells and thetiters of the virus were determined by plaque assay. Expression ofGM-CSF-G250 FP in Sf9 cells was determined by immunocytochemicalanalysis using anti-G250 mAb, anti-GM-CSF antibody (Genezyme, Cambridge,Mass.) and irrelevant Ab. Sf9 cells infected with pVL1392-Xy1Erecombinant virus (Pharmigen) and uninfected Sf9 cells were used asnegative control for FP expression and analysis. The viruses used forprotein production were isolated and amplified from a single plaque.Cell lysate was prepared from the Sf9 cells infected with viruses at MOIof 5 for three days with insect cell lysis buffer containing proteaseinhibitor cocktail (Pharmigen, San Diego, Calif.). Filtered lysate (0.22mm filter) was applied to a Ni²⁺-NTA agarose column with high affinityfor 6×His (Qiagen, Santa Clarita). After extensive washing of the column(50 mM Na-phosphate, 300 mM NaCl, 10% glycerol, pH 8.0), the fusionprotein was eluted stepwise column (50 mM Na-phosphate, 300 mM NaCl, 10%glycerol, pH 6.0) by increasing concentration of imidazole from 0.1M upto 0.5M. All purification steps were carried out at 4° C. Fractions wereanalyzed by Western blot using anti-GM-CSF antibody. The peak fractionswere combined, dialyzed and re-applied to an Ni^(2±)-NTA agarose columnfor repeated purification. Fractions containing FP were pooled, dialyzedand further applied to a FPLC column containing SP Sepharose, HighPerformance (Amersham Pharmacla Biotech, Piscataway, N.J.). Fusionprotein was eluted with an increasing salt gradient from 50 mM to 1MNaCl in buffer X (20 mM Tris, 1 mM EDTA, 10% Glycerol) and the fractionscontaining FP were pooled, dialyzed and sterilized through 0.2 m filter.The Coomassie blue and silver stains were used to analyze the purity ofGM-CSF-G250 fusion protein. The protein concentration was determined byBio-Rad Dc Protein Assay (Bio-Rad, Hercules, Calif. 94547).

GM-C SF Dependent Proliferation Assay

The biological activity of the GM-SF component of the FP was determinedby measuring the proliferation of GM-SF dependent TF-cells (Kitamura etal. (1989) J. Cellu. Physiol., 140: 323-334) in the presence of FP. TF-1cells were seeded in 96-well plates in triplicate in culture medium(RPMI medium+10% FBS) at the concentration of 2×10⁴ cells/wellcontaining titrated concentration of FP or the corresponding amount ofrecombinant human GM-CSF (rh-GM-CSF). Cultures were incubated for 5 daysand ³H thymidine (0.1 mCi/well) was added 12 h prior to harvest. Theincorporated ³H-thymidine was measured by scintillation counting with a0 counter.

Phenotypic Analysis of DC by Fluorescence Activated Cell Sorting (FACS)

The phenotype of DC generated from both adherent and bulk PBMC wasdetermined by two-color immunofluorescence staining as described inHinkel et al. (2000) J. Immunother., 23: 83-93. Both adherent PBMC andnon-fractionated bulk PBMC were cultured with 1000 U/ml of IL-4 pluseither GM-CSF (800 U/ml) or FP (0.34 mg/ml) for 7 days and the identityof DC was determined. Cell cultures (1×10⁵ cells) were re-suspended in50 ml FACS buffer (PBS, 2% new born calf serum, 0.1% sodium azide) andincubated with 10 ml of the appropriate fluorescein isothiocyanate(FITC) or phycoerythrine (PE) labeled monoclonal antibodies for 30 minat 4° C. After staining, cells were washed twice with PBS andre-suspended in 200 ml FACS buffer plus 200 ml paraformaldehyde 2%. Fiveto ten thousand events per sample were acquired on a Becton-DickinsonFACScan II flow cytometer that simultaneously acquires forward (FSC) andside scatter (SSC), as well as FL1 (FITC) and FL2 (PE) data, andanalyzed utilizing the CellQuest Software (Becton-Dickinson, San Jose,Calif.).

Settings for all parameters were optimized at the initiation of thestudy and were maintained constant throughout all subsequent analyses.DC population in bulk PBMC culture was gated based on their size andgranularity. In all samples the position of quadrant cursors wasdetermined by setting them on samples stained with the appropriatedisotype control antibody. The following antibodies were employed forcharacterization of the DCs phenotype: Anti-CD86 (B7-2; PharMingen,San-Diego, Calif.), Anti-CD40 (Caltag, Burlingame, Calif.), anti-HLAclass I (W6/32, ATCC HB95), anti-HLA-DR (Immunocytometry System; BectonDickinson, Mountain View, Calif.), anti-CD14 (Catlag laboratories, SanFrancisco, Calif.) and isotype control IgG1/IgG2a (Beckton Dickinson).The CD83⁺ surface marker was used to delineate the maturation of DC. Inorder to discriminate DC (CD83⁺CD19⁻) from activated B cells(CD83⁺CD19⁺), dual color staining utilizing CD19FITC and CD83PE(Immunotech, Marseille, France), was performed.

Semi-Quantitative Reverse Transcriptase-Polymerase Chain Reaction(RT-PCR) Analysis of Cytokine Profile in PBMC.

Total RNA was extracted from PBMC treated with FP (2.7 mg/107 cells) forvarious time intervals up to 24 hr at 37° C., using acid guanidineisothiocyanate-phenol-chloroform extraction. Reverse transcription ofmessenger RNA into cDNA was carried out by incubating titrated RNA withAMV reverse transcriptase, primer oligo (dT), dNTP, and RNAse inhibitorat 42° C. for 1 hour. One ml of each cDNA sample was amplified utilizingPCR in a total volume of 25 ml, (30 ng [³²P]-5′-oligonucleotide, 100 ng3′-oligonucleotide primer, 2.5 ml modified 10×PCR buffer, 1.25 units Taqpolymerase, and autoclaved double distilled water to a volume of 25 ml).The PCR mixture was amplified for 25 cycles in a DNA Thermocycler(Perkin-Elmer, Norwalk, Conn.). Each cycle consisted of denaturation at94° C. for one minute and annealing/extension at 65° C. for 2 minutes.The 32P-labeled PCR products were then visualized directly viaacrylamide gel electrophoresis and autoradiography and then quantitatedby excision of bands and subsequent scintillation counting.

The signal intensity of each amplified product was calibrated to itscorresponding β-actin mRNA expression as an internal control forquantitation of expression levels. In addition, quantitative analysiswas further elucidated by a serial dilution of mRNA (1:3, 1:10, 1:30 and1:300) and co-amplification of β-actin and GM-CSF mRNA. The sequences ofthe oligonucleotide primer pairs are as follows: β-actin: 5′-CAA CTC CATCAT GAA GTG TGA C-3′ (SEQ ID NO:11), 3′-CCA CAC GGA GTA CTT GCG CTC-5′(SEQ ID NO:12); GM-CSF: 5′-CCA TGA TGG CCA GCC ACT AC-3′ (SEQ ID NO:13),3′-CTT GTT TCA TGA GAG AGC AGC-5′ (SEQ ID NO:14); TNF-α: 5′-TCT CGA ACCCCG AGT GAC AA-3′ (SEQ ID NO:15), 3′-TAC GAC GGC AAG GAT TAC ATC-5′ (SEQID NO:16); IFN-γ: 5′-ATG AAA TAT ACA AGT TAT ATC TTG GCT TT-3′ (SEQ IDNO:17), 3′-ATG CTC TTC GAC CTC GAA ACA GCA T-5′ (SEQ ID NO:18); IL-2:5′-GGA ATT AAT AAT TAC AAG AAT CCC-3′ (SEQ ID NO:19), 3′-GTT TCA GAT CCCCTT TAG TTC CAG-5′ (SEQ ID NO:20); IL-4: 5′-CTT CCC CCT CTG TTC TTCCT-3′ (SEQ ID NO:21), 3′-TTC CTG TCG AGC CGT TTC AG-5′ (SEQ ID NO:22).

Immunomodulation of PBMC with Fusion Protein

Fresh isolated PBMC from patients with RCC expressing G250 were culturedin RPMI 1640 medium supplemented with 10% autologous serum. Variousschedules of immunomodulatory protocols of PBMC cultures with FP werecarried out as described in Table 3 and FIG. 10. The growth of PBMC wasdetermined by cell count, and the cytolytic activity of PBMCs wasassayed for different targets in a prolonged 18-hour chromium-51 (51Cr)release assay. Five thousand 51 Cr-labeled target cells per well wereseeded in a 96-well microtiter plate (Costar, Cambridge, Mass.) andmixed with PBMC yield several E/T ratios (40:1, 20:1, 10:1, and 5:1).Cytotoxicity was expressed as lytic units (LU) per 10⁶ effector cellswith lytic unit being defined as the number of effector cells thatinduce 30% lysis. T cell mediated and RCC specific cytotoxicity wasconfirmed by blocking assays in which targeted autologous tumor cellswere pretreated with anti-human leukocyte antigen (HLA) class I, classII, or PBMC were pretreated anti-CD3, anti-CD4, anti-CD8, or isotypecontrol antibody (Becton Dickinson) for 30 min at 4° C., before additionof cells to cytotoxicity culture plates. Spontaneous release of alltargets was equal or less than 20% of maximal release of 51-Cr release.The following target cells were used: autologous normal kidney cells(G250−), autologous RCC tumor cells (G250+), allogeneic RCC cells(G250+), allogeneic prostate cells (CL-1), and human fibroblast (hFb).

TABLE 3 Phenotypic Modulation of Bulk PBMC by Fusion Protein (FP) IL-2 +FP Pre- IL-2 + IL-4 + FP IL-2 + FP + IL-4 Phenotype cultured IL-2 FPIL-2 + FP FP IL-2 + FP CD56⁺CD3⁻ 25 13 11 4 9 1 CD5⁻CD3⁺ 46 47 70 84 8894 CD4⁺CD8⁻ 31 28 39 42 66 46 CD4⁺CD8⁺ 3 10 20 29 4 28 CD4⁻CD8⁺ 22 24 3124 22 25 CD3⁺TcR⁺ 40 45 72 69 79 96 CD3⁺CD25⁺ 19 43 61 54 17 86

Results

Generation of GM-CSF-G250 Fusion Protein from Baculovirus-Infected SF-9Cells.

Baculovirus expression technology and the 6×His affinity purificationsystem were used to generation GM-CSF-G250 FP as described above. Thesuccess of gene cloning and generation of recombinant baculovirus wasverified by the immunohistochemical staining of viruses infected Sf9cells using anti GM-CSF and anti-G250. Abundant G250 and GM-CSF proteinexpression were detected in Sf-9 cells that were infected withGM-CSF-G250 recombinant baculoviruses (FIG. 6A, top and middle panel),whereas no expression of GM-CSF or G250 was detected in non-infectedcells (FIG. 6A, bottom panel) or cells infected with pVL1392-Xy1Erecombinant viruses (data not shown). Western blot analysis was used toevaluate the efficiency of 6×His affinity tag in FP for Ni²⁺-NTAagarose. An expected 66-kDa band which detected with anti-GM-CSFappeared in the fractions collected from number 5 to number 25 with thepeak concentration at fraction 15 to 19 (FIG. 6B). The protein puritywas further improved by re-run of positive fractions through Ni²⁺-NTAagarose column and subjected to FPLC using SP Sepharose column. A majorsingle 66 kDa band was detected in SDS-PAGE analysis stained withcoomassie blue (FIG. 6C).

Purified GM-CSF-G250 Fusion Protein Retained GM-CSF Bioactivity

To determine whether the bioactivity of the GM-CSF was preserved in thepurified FP, the FP was analyzed for its ability to support theproliferation of a GM-CSF dependent cell line, TF-1. Serial dilutions ofFP were performed to span the effective concentration range. Theexperiments were conducted in parallel with recombinant GM-CSF. Theresults from the ³H-thymidine incorporation assay demonstrated that theFP could stimulate TF-1 cell growth with a biphasic dose dependentmanner (FIG. 7B). When compared to recombinant GM-CSF (FIG. 7A),comparable bioactivity was determined in the presence of FP withequivalent concentrations of GM-CSF in the range between 0-6.71 ng/ml(=0-30.2 ng/ml FP). In the presence of concentrations higher then 30.2ng/ml of FP, the growth induction of TF-1 by FP exceeded the growthinduction by recombinant GM-CSF by 1.3 fold (FIG. 7A, 7B).

Immunomodulatory Effect of Fusion Protein on Antigen Presenting Cells inPBMC Culture

In order to study how the FP could affect the development of DC, PBMCderived from patients with RCC were cultured in the presence of FP (0.34mg/ml) plus IL-4 (1000 U/ml) for 7 days and compared to that cultured inGM-CSF (800 U/ml) plus IL-4. FACS analysis revealed a high percentage oflarge granulocytes expressing B7-2⁺, CD40⁺ and HLA-DR⁺ in bothconditions whereas CD14⁺ cells were negligible (FIG. 8A). However, whencompared to dendritic cells cultured with recombinant cytokines, anenhanced expression of both HLA class I (mean relative linearfluorescence intensity=4830 vs 3215) and HLA class II (6890 vs 6290) wasdetected in the FP modulated DC cultures (FIG. 8B). In addition, therewas a three-fold increase of mature DC (CD83⁺CD19⁻) in FP modulated DCcultures (FIG. 8C). This observation was consistent in several bulk PBMCcultures derived from RCC patients (n=3) and healthy donors (n=2).Similar FP mediated immunomodulatory profile was also determined onconventional adherent DC cultures (data not shown). A lower efficiencyof DC differentiation was observed when DC were cultured in the presenceof FP alone without IL-4. A mix of CD14⁺ and CD14-B7-2⁺ cell populationwere determined on day 7 (data not shown).

Fusion Protein Induces Activation of Cytokine Genes in PBMC

To identify whether the fusion protein has a direct effect on theregulation of cytokine genes in PBMC, freshly isolated PBMC cells,derived from RCC patients, were treated with FP alone (2.7 mg/10⁷cells). The kinetics of cytokine gene activation was followed byanalysis of multiple cytokine mRNA expression through time course asindicated in FIG. 9. When compared to untreated PBMC, treatment ofuncultured PBMC with FP gradually enhanced GM-CSF, TNF-α, IFN-γ, IL-4,IL-2 mRNA expression with the peak level at 24 hr post treatment exceptIL-4. The peak of IL-4 mRNA expression was detected at 6 hr after thetreatment (FIG. 9).

Fusion Protein Induces T Cell Mediated and G250 Targeted Immune Responsein PBMC Cultures

Five immunomodulatory protocols with and without FP were tested andcompared in PBMC cultures. These culture conditions included 1) IL-2alone (40 IU/ml), 2) IL-2+FP (0.34 mg/ml) (re-stimulated weekly), 3)IL-2+IL-4 (1000 U/ml)+FP for one week then restimulated with FP+IL-2, 4)FP alone for one week then restimulated with FP+IL-2 and 5) FP+IL-4 forone week then re-stimulated with IL-2+FP. As indicated in FIG. 10(patient #1), among various immunomodulatory treatments tested, thecondition with pretreated PBMC with FP plus IL-4 for one week andsubsequently restimulated with IL-2 (40 ILT/ml) and FP weekly, showedthe highest growth expansion (6.0×) (FIG. 10A). A similar growth profilewith enhanced growth activity in this particular condition wasdetermined in another 3 PBMC cultures that were derived from patientswith RCC. In one particular patient (patient #11 who had a positivelymph node (LN), an enhanced cytotoxicity against LN derived tumortarget was determined in all four FP modulated PBMC cultures (3 cyclesof re-stimulation) when compared to the cytotoxicity against primarytumor target (FIG. 10B). Notably, this enhanced killing activitycorresponded to an eight-fold increase of G250 mRNA expression in LNderived RCC tumor, as determined by a semi-quantitative RT-PCR, whencompared to primary RCC cells (FIG. 10C). When LN tumor target cellswere pretreated with anti HLA class I (77%) or alternatively, effectorswere pretreated with anti CD3 (66%) or anti CD8 (55%) prior to theassay, RCC targeted cytotoxicity was markedly reduced. Whereas anti HLAclass II (33%) or anti CD4 (33%) treatment could lead only to a lesserinhibition of cytotoxicity (FIG. 10C). Although poor growth expansion(1.8×) was detected in the condition that pretreated PBMC with FP alonefor one week and re-stimulated with IL-2 plus FP, the highestcytotoxcity against both primary and LN derived RCC target was detectedwhen compared to other tested conditions (FIG. 10B).

To identify the phenotypic identity of FP modulated PBMC that possessanti-tumor activity, phenotypic analysis was performed on the day whencytotoxicity was determined. A markedly increased T cell population(70-94%) expressing T cell receptor (72-96%) was detected in all FPstimulated PBMC cultures, when compared to pre-cultured PBMC (46%) orPBMC cultured with IL-2 alone (47%) (Table 3). Notably, the T cellpopulation expressing the most IL-2 receptor (CD3⁺CD25⁺) (86%) wasdetermined to occur in the condition that pretreated cells with FP plusIL-4 prior to re-stimulation with IL-2 and FP. This also corresponded tothe greatest growth expansion in T cell population when compared to allother tested immunomodulatory protocols (FIG. 10A). Correspondingly,PBMC that were pretreated with FP for one week demonstrated a minimal Tcell population expressing the IL-2 receptor (19%) and demonstrating theleast growth expansion (Table 3 and FIG. 10A).

Replacement of FP with GM-CSF Abrogates the Selection of RCC TargetedCytotoxic T Cells

In order to confirm that the component of G250 in the FP is thedeterminant for the growth selection of CTL against RCC, cytotoxicityassay was performed with PBMC that were cultured in the presence ofGM-CSF and IL-4 for one week then continuously restimulated with IL-2and GM-CSF (800 U/ml). Minimal cytotoxicity against autologous RCC wasdetermined in all tested PBMC cultures without FP stimulation. Whereasthe corresponding PBMC cultures stimulated with FP showed an MHCrestricted, T cell mediated cytotoxicity against autologous RCC (FIG.11A) that expressed high level of G250 (data not shown). PredominantCD3⁺CD4⁺ cell population was detected in all three FP modulated PBMCcultures (68%, 74%, and 66%) that expressed antitumor activity, whencompared to CD3⁺CD8⁺ cell population (25%, 22%, and 30%) (FIG. 11B).Moreover, both Th1 and Th2 cytokine mRNA were detected in these FPmodulated PBMC cultures which included GM-CSF, TNF-α, IFN-γ, IL-2 andEL-4 (data not shown).

Discussion

Renal cell carcinoma (RCC) is responsive to immunotherapy. However, itis believed that no immune-based treatment protocol has been previouslyshown that would effectively eradicate tumor lesions in the majority ofpatients. It is believed that means of immune strategy, the type ofimmune activators used, the method of administration, and thepretreatment immune status of patients all could influence the ultimateimmune response in cancer patients that are treated with immune-basedtherapy. Therefore, an important issue for an effective cancer vaccineis the development of a potent adjuvant that can facilitate bothinduction and augmentation of an immune response with antitumoractivity. To achieve this, we proposed a chimeric construct consistingof G250 and GM-CSF. The demonstration of G250 expression in SF-9 cellsand GM-CSF bioactivity in the purified 66 kDa band of protein moleculeconfirmed the efficacy of the gene construct and the effectiveness ofthe selected protein purification method.

Antigen presentation by DC is important, not only for the induction ofprimary immune responses, but may also be important for the regulationof the type of T cell-mediated immune response (Banchereau et al. (2000)Ann. Rev. Immunol., 18: 767-811). We recently developed anon-fractionated bulk PBMC culture system for the study of thematuration and immunomodulatory function of CD14⁺ derived DC and theinteraction between the DC and co-cultured lymphocytes (Hinkel et al.(2000) J. Immunother., 23: 83-93). Using this system, antigen loadingcan be performed during the early culture period of PBMC in the presenceof GM-CSF and IL-4, when immature DC/monocytes can take up and processtumor antigen. As we have previously demonstrated that DC modulatedco-cultured lymphocytes in bulk PBMC culture can be further expanded toCTL by repetitive stimulation with low dose IL-2 and RCC tumor lysate.Likewise, direct treatment of bulk PBMC with IL-4 and FP not onlyinduced the differentiation of CD14⁺ cells into DC but also increasedthe maturation of DC when compared to DC generated in IL-4 and GM-CSF.This suggests that signaling pathway in DC maturation can be induced byFP stimulation (Rescigno et al. (1998) J. Exp. Med., 188: 2175-2180).Moreover, when compared to recombinant GM-CSF, up-regulated HLA antigenexpression was determined on FP modulated DC further indicating that DCare capable of internalizing, processing, and presenting FP through HLAantigens in DC. Whether the G250 was taken up by APC through GM-CSFreceptor internalization or via the G250 component remains to bedetermined.

It appears that preincubation of PBMC with FP and IL-4 prior to exposingIL-2 facilitates a better effector expansion. This may be explained ifexogenous IL-4 could synergize the FP for the mobilization of DCdifferentiation and maturation and subsequent presentation of theantigen peptides to the surrounding immune cells. Although a successfulCTL selection also could be achieved by other tested immunomodulatoryprotocols with FP, the growth expansion of CTL was not favorable. Thismay be partly associated with a “delayed” DC differentiation under thesub-optimal concentration of IL-4 (note: FP can induce IL-4 secretion byPBMC). Moreover, pre-exposure of IL-2 to a non-antigen stimulated PBMCusually results in the expansion of non-specific lymphokine activatedkiller cells with short-term killing activity (Roussel et al. (1990)Clin. Exp. Immunol., 82: 416-421). Recently, Huang et al. (1994)Science, 264: 961-965, demonstrated that even “immunogenic” tumors, suchas those modified to express co-stimulatory molecules, fail to stimulatethe immune system, unless functional APC are available to process andpresent the antigens. It thus appears that the most effective anticancer vaccine strategy should target manipulation of enhancing T cellpriming at the level of APC in patients.

That the replacement of FP with equivalent dose of recombinant GM-CSFabrogated the selection and propagation of RCC specific CTL suggeststhat activation and propagation of CTL is antigen (G250)-dependent.Whereas the GM-CSF has served as an effective adjuvant for antigenpresentation and amplification of T cell activity including cytokineresponse (Mach et al. (1999) Cancer Res., 60: 3239-3246; Pulendran etal. (1999) Proc. Natl. Acad. Sci., USA, 96: 1036-1041). Although FPinduced G250 targeted antitumor activity is mainly mediated by CD8′ Tcells, a predominant up-regulation of CD4′ T cells was detected in mostcultures. The FP mediated Th1 and Th2 cytokine release and enhancementof HLA class II expression in DC cells further suggests FP mediatedantitumor immunity may involve the priming of both CD4′ and CD8′ T cellsspecific for G250. The role of CD4′ T helper cells in this response maybe attributed to provide regulatory signals required for that priming ofMHC class I restricted CD8′ CTL (Mach et al. (1999) Cancer Res., 60:3239-3246).

Studies comparing the efficacy of various formulations of tumor vaccinesin parallel demonstrated that the use of DC transfected with DNA codingfor TAA is superior to peptide-pulsed DC and naked DNA based vaccine foreliciting both antigen-specific CD8 and CD4 T cell response (Yang et al.(1999) Intl. J. Cancer, 83: 532-540). This observation indicates thatcomputer predicted peptides might not be naturally processed andpresented on the tumor cells surface for the recognition by peptidereactive T cells. Thus, some in-vitro peptide-reactive T cells couldonly lyse peptide pulsed cell targets but not tumor cells expressing theentire tumor antigen (Vissers et al. (1999) Cancer Res., 59: 5554-5559;Rammensee et al. (1993) Annual Rev. Immunol., 11: 213-244). Likewise, apeptide-based vaccine could effectively elicit expansion of vaccinespecific T cells in PBMC of cancer patients, but such response was notassociated with a clinical tumor regression (Lee et al. (1999) JImmunol, 163: 6292-6300). Therefore, immunization with the currentconstruct of whole G250 antigen may have the advantage over the peptidesfor the presentation of multiple, or unidentified epitopes inassociation with MHC class I and class II molecules by APC. On the basisof the potency and specificity of the GM-CSF-G250 fusion protein in theactivation of G250-reactive T cells with antitumor activity, our dataindicate that vaccination with GM-CSF-G250 FP will provide therapeuticimpact for the treatment of advanced kidney cancer.

Example 3 Generation of the Mammalian Expression Vector pCEP4-GMCSF-G250

i) Amplification of the Recombinant Gene GMCSF-G250 without the his Tagand Cloning into pGEM-T.

pVL1393-GMCSF-G250 (His tag) was used as a template in a PCR reactionthat was carried out using primers designed to introduce a KpnI beforethe start codon of the GMCSF (5′ primer) and a XhoI site after the stopcodon (3′ primer). In addition, the 3′ primer was designed to eliminatethe poly-Histidine coding sequence previously introduced for detectionand purification purposes. A high fidelity amplification system (ExpandHigh Fidelity System, Boehringer-Mannheim) was used to avoid mutationsin the PCR product, which was directly cloned into pGEM-T vector(Promega), a convenient vector for further sequencing and cloning steps,resulting in pGEMT-GMCSF-G250. Completely sequencing of the GMCSF-G250gene revealed no mutation and the expected absence of the poly-histidinecoding sequence.

ii) Cloning of the GMCSF-G250 into the Mammalian Expression VectorpCEP4.

pCEP4 is an episomal vector mammalian expression vector that uses thecytomegalovirus (CMV) immediate early enhancer/promoter for high leveltranscription of recombinant genes inserted into the multiple cloningsites and also carries the hygromycin B resistance gene for stableselection in transfected cells. Subcloning of GMCSF-G250 into pCEP4 wascarried out with a digestion of vectors pGEMT-GMCSF-G250 and pCEP4 withrestriction enzymes KpnI and XhoI and further gel purification andligation of the resulting linearized pCEP4 and GMCSF-G250. The newplasmid pCEP4-GMCSF-G250 (FIG. 12) contained the recombinant gene in theproper orientation as expected. A SalI digestion of pCEP4-GMCSF-G250released the complete expression cassette CMVpromoter-gene-polyadenylation signal (3.7 kb, FIG. 13) that can becloned into the E1 and E3 deleted adenovirus or gutless adenovirusbackbone for generation of fusion gene recombinant adenovirus. Thesefusion gene recombinant viruses can be used as a virus-form to immunizepatients directly or alternatively, to infected RCC cells or DC togenerate kidney cancer vaccine for the direct immunization of patients.Alternatively, defined RCC cell lines can be stably transfected withpCEP4-GMCSF-G250 and used as RCC tumor vaccine. These various types ofG250-GM-CSF vaccine formulations also can be used as an in-vitroimmunostimulant for activation and propagation of G250 targeted CTL fromPBMC or TIL cultures, which derived from patients with RCC then,re-infuses these CTL back to patients as an adoptive immunotherapy.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1-17. (canceled)
 18. A nucleic acid encoding a fusion protein comprisinga G250 kidney cancer specific antigen attached to a granulocytemacrophage colony stimulating factor (GM-CSF).
 19. The nucleic acid ofclaim 18, wherein said nucleic acid is a deoxyribonucleic acid (DNA).20. The nucleic acid of claim 18, wherein the G250 antigen is a humanG250 antigen and the GM-CSF is a human GM-CSF.
 21. The nucleic acid ofclaim 20, wherein the G250 antigen and the GM-CSF are joined by apeptide linker ranging in length from 2 to about 20 amino acids.
 22. Thenucleic acid of claim 21, wherein said peptide linker is -Arg-Arg-. 23.The nucleic acid of claim 22, wherein said nucleic acid encodes thepolypeptide of SEQ ID NO:
 1. 24. The nucleic acid of claim 22, whereinsaid nucleic acid comprises the nucleic acid of SEQ ID NO:
 2. 25. Thenucleic acid of claim 20, wherein said nucleic acid is present in anexpression cassette.
 26. The nucleic acid of claim 20, wherein saidnucleic acid is present in a vector.
 27. (canceled)
 28. A host celltransfected with a nucleic acid comprising the nucleic acid of claim 18.29. The host cell of claim 28, wherein said host cell is a eukaryoticcell.
 30. (canceled)
 31. A method of producing an anti-tumor vaccine,said method comprising: culturing a cell transfected with a nucleic acidcomprising the nucleic acid of claim 18 under conditions where saidnucleic expresses a G250-GM-CSF fusion protein; and recovering saidfusion protein. 32-77. (canceled)
 78. A method of inhibiting theproliferation or growth of a transformed renal cell that bears a G250antigen, said method comprising: removing an immune cell from amammalian host; activating said immune cell by contacting said cell witha protein comprising a renal cell carcinoma specific antigen (G250)attached to a granulocyte macrophage colony stimulating factor (GM-CSF)or a fragment thereof; optionally expanding the activated cell; andinfusing the activated cell into an organism containing a transformedrenal cell bearing a G250 antigen.
 79. The method of claim 78, whereinsaid removing comprises obtaining peripheral blood lymphocytes or TILsfrom said mammalian host.
 80. The method of claim 79, wherein saidinfusing comprises infusing the activated cells into the host from whichthe immune cell was removed.
 81. The method of claim 79, wherein saidimmune cell is selected from the group consisting of a dendritic cell,an antigen presenting cell, a B lymphocyte, a T-cell, and a tumorinfiltrating lymphocyte.
 82. A method of treating an individual having arenal cell cancer, said method comprising: (a) sensitizing antigenpresenting cells in vitro with a sensitizing-effective amount of achimeric fusion protein comprising a renal cell carcinoma specificantigen (G250) attached to a granulocyte macrophage colony stimulatingfactor (GM-CSF); and (b) administering to an individual having saidrenal cell cancer or metastasis a therapeutically effective amount ofthe sensitized antigen presenting cells.
 83. The method of claim 82,wherein the antigen presenting cells are autologous to the individual orare MHC matched allogenic dendritic cells.
 84. The method of claim 82,wherein said sensitizing comprises contacting cells selected from thegroup consisting of peripheral blood lymphocytes, monocytes,fibroblasts, TILs, and dendritic cells with said chimeric fusionprotein.
 85. The method of claim 82, wherein said sensitizing comprisestransfecting dendritic cells or RCCs with a nucleic acid encoding saidchimeric fusion protein.